Bos indicus) with improved (Bos taurus) breeds gained momentum and economic relevance in several countries to increase milk production. While production performance of the crossbred offspring is high due to hybrid vigor, they suffer from a high incidence of reproductive problems. Specifically, the crossbred males suffer from serious forms of subfertility/infertility, which can have a significant effect because semen from a single male is used to breed several thousand females. During the last two decades, attempts have been made to understand the probable reasons for infertility in crossbred bulls. Published evidence indicates that testicular cytology indices, hormonal concentrations, sperm phenotypic characteristics and seminal plasma composition were altered in crossbred compared to purebred males. A few recent studies compared crossbred bull semen with purebred bull semen using genomics, transcriptomics, proteomics and metabolomics; molecules potentially associated with subfertility/infertility in crossbred bulls were identified. Nevertheless, the precise reason behind the poor quality of semen and high incidence of sub-fertility/infertility in crossbred bulls are not yet well defined. To identify the underlying etiology for infertility in crossbred bulls, a thorough understanding of the magnitude of the problem and an overview of the prior art is needed; however, such systematically reviewed information is not available. Therefore, the primary focus of this review is to compile and analyze earlier findings on crossbred bull fertility/infertility. In addition, the differences between purebred and crossbred males in terms of testicular composition, sperm phenotypic characteristics, molecular composition, environmental influence and other details are described; future prospects for research on crossbred males are also outlined.Crossbreeding of indigenous cattle ( Bos taurus and Bos indicus is being practiced in several countries  around the world are engaged in milk production, mainly in developing countries . The daiountries  resultinountries . Crossbrountries . Since tountries . Even thountries . Female ountries . Globallountries .Infertility in a bull is defined as the inability to achieve pregnancies, whereas sub-fertile bulls  delay conception, prolong the calving season, reduce calf weaning weights, and increase the numbers of females culled, thereby resulting in economic losses and threatening the sustainability of a livestock operation . Breed vBos taurus male with Bos indicus female, although not sterile, show increased incidences of sub-fertility and/or infertility compared to their parents (Bos indicus) and European taurine cattle (Bos taurus) exhibit several morphological and physiological differences. At chromosome level, the Y-chromosome in Bos taurus is submetacentric while that in Bos indicus is acrocentric. Therefore, it was proposed that the lower fertility in zebu crosses with European cattle could be due to small deletions or position changes between the synapse region of the X and Y chromosomes, or to alterations in genes participating in the regulation of reproduction  are always sterile. The male progeny born via crossbreeding of a  parents . Despiteoduction . With thoduction . While toduction  indicateDespite these fertility problems, crossbreeding is commonly adopted to improve the milk productivity of indicine and non-descript cattle; the reason for the higher incidence of sub-fertility or poor semen quality in crossbred bulls compared to purebred bulls is not fully understood. Understanding the reason behind infertility/subfertility in crossbred bull will assist development of strategies to improve crossbred bull reproduction. Therefore, in this review, we compiled and analyzed earlier findings on crossbred bull fertility/infertility. In addition, the differences between purebred and crossbred males in terms of testicular composition, sperm phenotypic characteristics, molecular composition, environmental influence, and other details are described and future prospects for research on crossbred males are also outlined.B. indicus × B. taurus (22%) bulls than purebred Bos indicus (13%) and Bos taurus (15%) bulls  bulls . More th%) bulls . Among t%) bulls . Ejacula%) bulls . EjaculaEjaculate quality in crossbred bulls is also influenced by the level of exotic inheritance , althougThe incidence of sub-fertility/infertility in crossbred bulls is high compared to purebred bulls. Sire conception rate was higher in indigenous bulls , compared to Holstein Friesian crossbred  and Jersey crossbred  bulls . AnotherHigher culling rates (40–70%) due to poor semen quality, poor sperm cryotolerance and sub-fertility/infertility were reported in crossbred bulls . In a stBos indicus males (Bos taurus (40%) and Bos indicus (29%)  . A signius (29%) . Singh eus (29%) .In an effort to understand the reasons behind the higher incidence of infertility/sub-fertility in crossbred males, several researchers studied the physiological and molecular differences between crossbred and purebred males. Some studies proposed axiomatic evidences at genetic , hormonaDuring spermiogenesis, chromatin compaction occurs by replacing histones with protamines; little or no cytoplasm remains in spermatozoa . TherefoPRM1 is an abundant transcript found in spermatozoa of crossbred bulls  based kits were developed  in purebred bulls compared to crossbred bulls  were upregulated, whereas genes involved in WNT pathway (involved in sperm motility initiation and inhibition of ubiquitination) were downregulated in crossbred testes. The genes involved in steroidogenesis, including CYP17A1 gene  were downregulated in crossbred bull testis. Genes associated with steroidogenesis are downregulated in crossbred bull testis compared to purebred testis. Downregulated steroidogenesis-related genes and their involvement as a group in different biological process, molecular function and pathway are shown in An appropriate molecular environment in the testis is required for proper spermatogenesis. In a recent study, the testicular transcripts related to sperm function and fertilizing potential were altered in crossbred bull testis compared to purebred bull testis . This stIn a study by Successful reproduction depends upon an optimal endocrine milieu. Development of testis, puberty, sexual maturity and spermatogenesis require certain levels of reproductive hormones, mainly follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone . AlthougBos taurus × Bos indicus) cattle  cattle . The X a) cattle . This PA) cattle . Synapse) cattle .Inter-species breeding can cause structural and/or molecular/genetic discrepancy between the PAR of X and Y chromosome in the offspring and interferes in the usual synapse and recombination during male meiosis, resulting in infertility . For insBos taurus with Bos indicus, because the Y chromosome of Bos taurus is submetacentric whereas in Bos indicus it is acrocentric with visible p-arms. The Y chromosomal arms are of different sizes in Bos indicus and Bos taurus. Furthermore, the location of PAR on the Y chromosome differs as it is located on the short arm of Bos taurus Y chromosome but on the long arm of Bos indicus Y chromosome. The difference between the Y chromosome of Bos taurus × Bos indicus is due to pericentric inversion or centromere transposition. The Crossbred (Bos taurus × Bos indicus) bulls have a submetacentric Y chromosome (Bos taurus and Bos indicus (which have different Y chromosome morphology and a different location of PAR in the Y chromosome) can result in structural and/or molecular differences between the PAR of X and Y chromosome in the male offspring. Thus, normal pairing and recombination between the PAR of X and Y chromosome during male meiosis would be disrupted. This may eventually lead to improper spermatogenesis and infertility in crossbred bulls. Although the location of PAR on the short or long arm does not completely block the pairing between X and Y chromosomes, it interrupts the success of X-Y pairing by having genetic consequences in structural chromosomal reorganization. For instance, isochromosome formation results in duplication or complete deletion in one of the sex chromosomal regions (The same reasoning can be speculated for the progeny born out of the crossbreeding romosome . As ment regions .SRY), DEAD box polypeptide 3-Y chromosome (DDX3Y) and Testis-specific protein on Y chromosome (TSPY)] of Holstein Friesian crossbred bulls compared to Sahiwal (Bos indicus) bulls. Furthermore, PRAMEY , TSPY , ZNF280BY , and HSFY  in the Y chromosome of F1 hybrid cattle-yak bulls than in cattle bulls. However, in-depth studies involving a large number of different purebred and crossbred males are required to understand the effect of a discrepancy between the PAR of X and Y chromosome on male fertility.Disparity between sex chromosomes due to species hybridization or crossbreeding may also lead to deletions and duplication of genes in the offspring. This is supported by the findings of Bos taurus with Bos indicus might eventually lead to improper spermatogenesis and infertility in crossbred bulls because of differences in the Y chromosome. The Y chromosome of Bos taurus is submetacentric, whereas in Bos indicus it is acrocentric. Differences in location of PAR in the Y chromosome might disrupt the process of normal pairing and recombination between the PAR of X and Y chromosome during male meiosis. Information about the influence of the level of exotic blood on fertility in crossbred males is very limited. Schematic representation of the possible cellular and molecular alterations in crossbred testis and spermatozoa that might culminate in subfertility/infertility is given in Undoubtfully, crossbreeding of low-producing zebu cows with exotic bulls of high genetic merit has resulted in the production of superior genotypes with hybrid vigor and enhanced milk production efficiency. On the other hand, it is evident from the foregoing information that the magnitude of reproductive problems is higher in crossbred bulls compared to purebred bulls. Until recently, little research had been done to identify the underlying etiological factors for this high incidence of infertility/sub-fertility in crossbred bulls. During the last decade, considerable research has been conducted; published information suggests alterations at the level of testis, spermatozoa, seminal plasma and male reproductive hormones, in crossbred bulls compared to purebred bulls. It is also evident that crossbreeding of Bos taurus, Bos indicus, and their crossbred male offspring should be studied to understand the origin of these reproductive problems in crossbred males. Expression of genes such as DDX3Y, RINGO, and SPATA7 and metabolites such as hypotaurine, L-malic acid, selenocysteine, D-cysteine, and chondroitin 4-sulfate are to be studied further to determine their role in crossbred male reproduction. Testicular transcriptomic study indicated an inability of the crossbred testis to maintain protein stability and steroidogenesis, which could be the pressure point for reproductive problems in crossbred bulls. Therefore, research to understand and improve protein stability and steroidogenesis in crossbred bulls will be important in the future. In addition, tailored freezing techniques and specific extenders need to be tested because poor freezability of spermatozoa is an important problem in crossbred bulls. Although sperm mRNAs (messenger RNA) were studied to some extent in crossbred bulls, the other RNAs such as transfer RNA (tRNA), ribosomal RNA (rRNA), long non-coding RNA (lncRNA), mitochondrial RNA (mt-RNA), small non-coding RNA (sncRNA), small nuclear RNA (snRNA), and small nucleolar RNA (snoRNA) need to be investigated to broaden our understanding about crossbred bull fertility. Studies involving microRNA, small interfering RNA, their intracellular delivery and target genes in crossbred bulls are particularly warranted. Furthermore, use of technologies such as gene transfer, editing, slicing and knockout may be useful in future to understand and improve the fertility in crossbred bulls.Detailed and large-scale studies involving crossbred males with different levels of exotic inheritance are required to have a clear understanding about infertility/sub-fertility in crossbred bulls. Genes in the pseudo autosomal regions of X and Y chromosome of AK, KE, and TD conceptualized the review. All authors were involved in literature review and development of the manuscript, and approved the manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rubella virus (RuV) is the causative agent of rubella (“German measles”) and remains a global health concern. Until recently, RuV was the only known member of the genus Rubivirus and the only virus species classified within the Matonaviridae family of positive-sense RNA viruses. Recently, two new rubella-like matonaviruses, Rustrela virus and Ruhugu virus, have been identified in several mammalian species, along with more divergent viruses in fish and reptiles. To screen for the presence of additional novel rubella-like viruses, we mined published transcriptome data using genome sequences from Rubella, Rustrela, and Ruhugu viruses as baits. From this, we identified a novel rubella-like virus in a transcriptome of Tetronarce californica—order Torpediniformes (Pacific electric ray)—that is more closely related to mammalian Rustrela virus than to the divergent fish matonavirus and indicative of a complex pattern of cross-species virus transmission. Analysis of host reads confirmed that the sample analysed was indeed from a Pacific electric ray, and two other viruses identified in this animal, from the Arenaviridae and Reoviridae, grouped with other fish viruses. These findings indicate that the evolutionary history of the Matonaviridae is more complex than previously thought and highlights the vast number of viruses that remain undiscovered. Rubella virus (RuV) (Matonaviridae: Rubivirus) is a single-stranded positive-sense RNA virus [NA virus . It is bNA virus . On occaNA virus ,4. DespiNA virus , they arNA virus . RuV, thNA virus .Rubivirus, the only genus in the family Matonaviridae [Guangdong Chinese water snake rubivirus, was discovered via a large virological survey of vertebrates in China and is highly divergent from RuV, only sharing around 34% amino acid identity [Tiger flathead matonavirus was identified through a metatranscriptomic exploration of Australian marine fish [Guangdong Chinese water snake rubivirus, forms a sister clade to RuV. Even more recently, two novel rubella-like virus species were described: Rustrela virus, identified in several mammalian species in, and in close proximity to, a zoo in Germany, and Ruhugu virus from bats in Uganda [The disease rubella was first described in 1814 [aviridae . This piidentity . Similarine fish , and, aln Uganda . These vRubella, Ruhugu, and Rustrela viruses.The popularisation of metagenomic methods to identify novel viruses within tissue or environmental samples has enabled their rapid discovery and provided important insights into viral diversity and evolution ,14. HereRustrela virus , Ruhugu virus (MN547623), and RuV strain 1A (KU958641) were screened against transcriptome assemblies available in NCBI’s TSA database  using the online translated Basic Local Alignment Search Tool (tBLASTn) [Homo sapiens (taxonomic identifier: 9606), and utilised the BLOSUM45 matrix to increase the chance of finding highly divergent viruses. Putative viral sequences were then queried using the DIAMOND BLASTx (v.2.02.2) [To identify the presence of novel rubella-like viruses in vertebrates, translated genome sequences of tBLASTn) . Searche.2.02.2)  algorithTo recover further fragments of the novel matonavirus genome and screen for other viruses in the Pacific electric ray transcriptome (see Results), raw sequencing reads were downloaded from the NCBI Short Read Archive (SRA) (Bioproject: PRJNA322346). These reads were first quality trimmed and then assembled de novo using Trinity RNA-Seq (v2.11) with the “trimmomatic” flag option . AssemblVirus abundance, expressed as the standardised total number of raw viral sequencing reads that comprise a given contig, was calculated using the “align and estimate” module available for Trinity RNA-seq with the “prep_reference” flag enabled, RNA-seq by Expectation-Maximization (RSEM)  as the mA range of virus genomes representative of each virus family and sub-family under consideration, and for a range of host species, were retrieved from GenBank for phylogenetic analysis. The amino acid sequences of previously described viruses were aligned with those generated here with Multiple Alignment using Fast Fourier Transform (MAFFT) (v.7.4)  employinT. californica viruses described were named with “Tetronarce” preceding their respective family name to signify their electric ray host.The new Rustrela and Ruhugu virus sequences used for data mining in the project are available under Bioproject PRJNA576343. The raw T. californica sequence reads are available at Bioproject PRJNA322346. All other publicly available sequences used in analyses can be accessed via their corresponding accession codes (see relevant figures). Consensus sequences of the new viruses identified in this study are available on GenBank under accession numbers BK014632, BK014633, BK014634 and BK014635.The Tetronarce matonavirus. Screening of Rustrela, Ruhugu, and Rubella 1A virus sequences against the TSA database revealed matches to a contig of 3006 nucleotides (1001 amino acids) from a transcriptome of the electric organ of a female Pacific electric ray  [Rubella virus  from a divergent rubi-like virus that we tentatively named fornica) , rangingTetronarce matonavirus, we de novo assembled the raw sequencing reads from the Pacific electric ray sample. Accordingly, transcripts of a non-structural polyprotein, the p90 (RdRp) protein (815 amino acids in length in RuV), were recovered. By mapping contigs to the ruhugu virus non-structural polyprotein reference sequence, a continuous 399 amino acid section of the RdRp was identified and used for additional phylogenetic analysis. The RdRp fragment matched most closely to the RdRp of RuV  and a 615 amino acid segment of the segment 1 protein of a novel reovirus (Tetronarce reovirus). BLASTx searches indicated that the 753 amino acid Tetronarce arenavirus fragment was most closely related to salmon pescarenavirus 1 , followed by Tetronarce arenavirus (8.0 × 10−7), and then Tetronarce reovirus (2.7 × 10−7). The abundance of reads from the host gene, RPS13, was 2.4 × 10−4.We estimated the standardised abundances of the three novel viruses in comparison to the stably expressed host gene, 40S ribosomal protein S13 (RPS13) a. RPS13 Matonaviridae, as well as the RdRp (L segment) of Arenaviridae  and forming an ingroup to Ruhugu virus (found in bats). Strikingly, therefore, Tetronarce matonavirus does not group with the only other fish  virus in this family, Tiger flathead matonavirus, and the evolutionary history of this virus family does not follow strict virus–host co-divergence.For phylogenetic analysis of Tetronarce arenavirus falls within a group of viruses that have been identified in other fish host species, including Big eyed perch arenavirus and Wenling frogfish arenavirus-2. Unlike the Reoviridae and Matonaviridae phylogenetic trees, all fish arenaviruses identified to date form a distinct fish arenavirus clade.In contrast, Reoviridae comprises two sub-families (Sedoreovirinae and Spinareovirinae). Reoviruses have previously been identified in fish (Spinareovirinae). The Tetronarce reovirus and its closest match in the NCBI database  known to infect various mammalian hosts [African horse sickness virus (AHSV) and Equine encephalosis virus (EEV), and the more recently discovered snake-associated Letea virus [The  in fish e, althouan hosts , includiea virus .Tetronarce matonavirus , exhibited little conservation of the E1 protein and other structural proteins overall. There is clear conservation in regions corresponding to epitopes N2–N4 between the mammalian viruses and the electric ray virus, including a glycine residue in N4 conserved across all five viruses in the alignment  from a divergent matonavirus in a transcriptome of a Pacific electric ray. Additionally, partial RdRp transcripts of other novel viruses within the Tetronarce matonavirus identified here exhibited clear sequence identity to RuV, as well as to the Rustrela and Ruhugu viruses recently discovered in other mammals. High levels of sequence conservation within the E1 structural protein have been previously shown among RuV, Rustrela virus, and Ruhugu virus [Tetronarce matonavirus shares comparable levels of amino acid conservation to mammalian matonaviruses in this region of E1, whereas the other matonavirus from a non-mammalian host (Guangdong Chinese water snake virus) does not. However, we were unable to recover a structural polyprotein from Tiger flathead matonavirus to incorporate it into our analysis. The non-structural fragment of the novel virus also contains a highly conserved GDD motif within the RdRp, which is highly conserved across many RNA viruses and is essential for efficient replication [The novel gu virus , includigu virus ,32. Tetrlication .Tetronarce matonavirus is more closely related to Rustrela virus than to the other, more divergent, fish matonavirus, and hence is indicative of a complex history of cross-species transmission events. Such a conclusion is not dependent on the position of the tree root. Given that this phylogenetic position is somewhat paradoxical, it is important to exclude sample contamination, or that the Tetronarce matonavirus was in fact obtained from a component of the diet of T. californica. Importantly, no significant non-fish matches were identified in the T. californica transcriptome library, with most host reads producing matches to T. californica or related fish species. Similarly, that the novel arenavirus and reovirus identified were most closely related to previously documented fish-associated viruses, rather than those from mammals, again suggests that the unusual phylogenetic position of Tetronarce matonavirus is bona fide. Hence, these data are indicative of a complex history of cross-species transmission of viruses in the Matonaviridae, potentially involving cross-species transmission events among different vertebrate classes. Evidence of such expansive host-jumping events has been observed in the evolution of other virus families, such as hepatitis B viruses in the Hepadnaviridae, in which a number of fish viruses group closely with those identified in mammals [Matonaviridae with viruses from such a broad host range suggests this process has also played an important role in the evolutionary history of these viruses. Further screening of transcriptomes will also be paramount in solidifying Tetronarce matonavirus’ position in the Matonaviridae tree by revealing additional evolutionary links.Of particular note was that phylogenetic analysis suggested that  mammals . Indeed, mammals , and theTetronarce reovirus—was also identified in our analysis. The majority of reoviruses found in fish fall within the sub-family Spinareovirinae. In contrast, Tetronarce reovirus appears to be one of two viruses currently known that fall within the Sedoreovirinae sub-family that include vector-borne orbiviruses, such as AHSV and EEV, that cause fatal diseases in equines [Letea virus [Tetronarce reovirus is most clearly related to another fish-associated reovirus—Wenling scaldfish reovirus. Similarly, the Arenaviridae has also been recently expanded, with a host range that was previously thought to be limited to rodents and humans now expanded to include other warm- and cold-blooded vertebrates [Tetronarce arenavirus—clusters with a group of fish and amphibian viruses.A potentially new reovirus— equines ,37. Howeea virus . It is atebrates . The novMatonaviridae, as well as a relatively high viral abundance compared with a stably expressed host gene, is highly suggestive of the existence of a novel rubi-like virus in T. californica. While screening of annotated contigs and the existence of two other potentially novel viruses sharing sequence identity to other fish viruses in the assembled transcriptome suggest the Pacific electric ray to be the true host of these viruses, further surveying of this species for viral genomes would be beneficial to further understanding the origin and emergence of matonaviruses like RuV.The recovery of a complete structural polyprotein and a partial RdRp, both with conserved amino acid sequences and motifs present across Matonaviridae and suggest a more complex evolutionary history than previously suspected. More broadly, this study demonstrates the value of performing extensive transcriptome sequencing and screening of a wide range of potential hosts to identify animal relatives of viruses of significant health concern and to characterise more of the global virosphere.Overall, these findings expand the currently established host range of


Bunyavirales. This article overviews the major plant viruses that are part of the order, i.e., orthospoviruses (Tospoviridae), tenuiviruses (Phenuiviridae), and emaraviruses (Fimoviridae), and provides updates on the more recent ongoing research. Features shared with the animal-infecting counterparts are mentioned, however, special attention is given to their adaptation to plant hosts and vector transmission, including intra/intercellular trafficking and viral counter defense to antiviral RNAi.Negative-strand (-) RNA viruses (NSVs) comprise a large and diverse group of viruses that are generally divided in those with non-segmented and those with segmented genomes. Whereas most NSVs infect animals and humans, the smaller group of the plant-infecting counterparts is expanding, with many causing devastating diseases worldwide, affecting a large number of major bulk and high-value food crops. In 2018, the taxonomy of segmented NSVs faced a major reorganization with the establishment of the order  Mononegavirales) and those with segmented genomes. Plant viruses, although being a relative minority of NSVs, have representatives in both groups within the families Rhabdoviridae and Bunyaviridae, and several floating genera . The first overview of the plant-infecting viruses among the NSVs was published a decade ago . Th. Th1]. Tiviridae . All comiviridae . Tenuiviiviridae . Along tiviridae . Earliermi (UUK) , but alsmi (UUK) . During mi (UUK) . If and mi (UUK) . A recenmi (UUK) .As with other viruses from the bunyavirales, tenuivirus proteins are expressed from subgenomic or near-genomic length viral mRNAs that are transcriptionally initiated by cap-snatching, similar to tospoviruses . To thi2+ signaling network involved in the regulation of ion homeostasis. RGSV-infected plants show a significant decrease of potassium content, whereas some RGSV symptoms mimic potassium deficiency [N. benthamiana that potentially could target eIF4A mRNA for regulation, causing leaf twisting and stunting [How plant viruses induce symptoms in their hosts has not been completely elucidated. Symptoms appear as consequence of infection due to the hijacking of important plant functions by viral components. For tenuiviruses, RGSV p5 protein is shown to interfere with the CBL–CIPK Caficiency . It has ficiency . RSV infficiency . Upon RGficiency . The RGSficiency . A regiostunting .Chloroplasts play an important role in virus infection, and virus-induced alteration of chloroplasts is often associated with photosynthesis defects and chlorotic symptoms ,87. GeneEmaravirus particles resemble those of orthotospoviruses, although they appear distinct on one point. In most emaraviruses, double-membrane-bound bodies (DMBs)/particles, ranging from 80 to 200 nm in diameter, have been observed d. WithinActinidia chlorotic ringspot-associated virus (AcCRAV) and Pigeon pea sterility mosaic virus (PPSMV-1) have only five RNA segments [Within the figure on genome organizations, the emaravirus HPWMV genome is taken as a reference, with eight genome segments , each ensegments ,92. AcroAs for tenuiviruses, RNA1 encodes the viral RdRp, RNA2 encodes the envelop GP precursor, RNA3 encodes the viral N protein, and RNA4 encodes the viral MP . The RdR201/D202D, predicted to yield two mature glycoproteins Gn (22.4 kDa) and Gc (51.6 kDa) [Juju yellow mosaic virus GP suggests that there are two protease cleavage sites, V25ES/SS and V218LA/DD. Processing of the precursor polyprotein produces three mature glycoproteins, Gn (22.3 kDa), Gs (3.0 kDa), and Gc (50.89 kDa) [23/K24V and VKA196/E197D and are predicted to produce Gn (19.8 kDa), Gs (2.6 kDa), and Gc (53.1 kDa) [Envelope glycoproteins Gn and Gc are derived from the GP precursor encoded by RNA2 . Among e1.6 kDa) . Analysi.89 kDa) . For AcC3.1 kDa) . Whether3.1 kDa)  is not kRNA3 encodes the N protein of 32–37 kDa, and can vary from 12 to 80% amino acid sequence identity between species . At the RNA4 encodes the MP of approximately 27 kDa. Studies using Raspberry leaf blotch virus (RLBV) and FMV showed that each P4 protein could rescue a movement-defective potato virus X that lacks a portion of the TGB1 movement protein ,107,108.Fimoviridae and share identity with the animal- and insect-infecting Peribunyaviridae and Cruliviridae and two genera of Phasmaviridae (Feravirus and Jonvirus) [de novo or involves endogenous priming.The 3′ and 5′ termini of all RNA segments are highly conserved and show inverted complementarity, thereby enabling the formation of a panhandle e and cononvirus) . The endonvirus) ,111,112.Bunyavirales. However, they encode one additional non-structural accessory protein  and their functions, and expression strategy to homologs from the animal-infecting members of the  segment ), in a cDuring the intra/intercellular trafficking, NSm interacts with ER chaperones from the DnaJ protein family , co-chapAlthough NSm assists in cell-to-cell movement of viral entity in a tubule-guided manner, it is also able to complement movement-deficient Tobacco mosaic virus (TMV) in cell-to-cell and long-distance movement ,124. TMVPeribunyaviridae and Phenuiviridae, is involved in the modulation of host antiviral defense responses. While in animal systems this involves antagonistic properties of interferon-induced defense responses and shut-off of host cell protein synthesis [The second non-structural protein , like tynthesis , in planynthesis  RNAs and thereby interfere in host gene regulation and plant development . This agMutation of a WG/GW motif, known to enable Argonaute (Ago) binding (the core component of RISC), compromises the ability of TSWV NSs to suppress local RNA silencing, but not systemic silencing, and implies that this mutant is still binding siRNAs and thereby prevents systemic silencing ,134,135.Watermelon silver mottle orthotospovirus (WSMV) indicated the importance of this domain in self-interaction and RNAi suppressor activity [Alanine substitution analysis of TSWV NSs showed the importance of the N terminal domain in RNA silencing suppression , while mactivity ,136, altactivity ,138.In agreement with its role in virulence, the NSs protein is able to restore the pathogenicity of suppression-deficient heterologous viruses and support systemic movement ,139,140.Capsicum chlorosis orthotospovirus (CaCV) has NSs been reported to also localize in the nucleus [In plants, NSs is localized in the cytoplasm where, depending on the isolate, it is found in fibrillar structures or paracrystalline arrays ,143. Onl nucleus , but itsSpodoptera frugiperda insect cells [Orthotospoviruses also replicate during their persistent transmission by thrips. After uptake by L1/L2 larval stages, viral proteins can be detected in the foregut, midgut, ligaments, and salivary gland tissues. Whereas in larvae the primary site of replication involves the midgut epithelium (and nearby muscle cells) and tubular salivary glands, this shifts towards the primary salivary glands in adults, and suggests tissue tropism changes with insect development. Within the salivary glands, accumulating amounts of N and NSs proteins are detected, and mature particles can be discerned secreted into the salivary gland ducts ,148,149.ct cells . Furtherct cells .Tenuiviruses encode several non-structural proteins with known or unknown function , of whicN. benthamiana, showing that this protein contributes to long-distance virus spread [N. benthamiana epidermal cells, P2 (fused with YFP) initially moves to the nucleolus, where it co-localizes with Cajal bodies [N. benthamiana cells. Fibrillarin depletion affects the nucleolar and cytoplasmic localization of p2-YFP fluorescence and abolishes systemic movement of RSV but not of several positive-stranded viruses [Besides cell-to-cell movement, the systemic infection of plants requires that viruses enter the sieve elements and spread through the phloem to infect new organs. Silencing of RSV p2 protein abolished systemic movement in s spread . Upon trl bodies . At latel bodies . P2 inte viruses . The mecA. thaliana, the protein can partially substitute for the function of dsRNA-binding domain (dsRBD) of AtDRB1/AtHYL1 during miRNA biogenesis. As a result, p3 induces the accumulation of several miRNAs, most of which target pivotal genes associated with development or pathogen resistance. Therefore, p3 is postulated to hijack OsDRB 1, a key component of the processing complex, for the biogenesis of miRNAs, and support virus infection and pathogenesis in rice [N. benthamiana interacts with p3 and mediates its degradation by the 26S proteasome, participating in plant defense against infection [N. benthamiana confers resistance to RSV, confirming the role of autophagy in suppressing RSV infection [Like nearly all plant viruses, tenuiviruses also encode proteins counteracting plant defenses, particularly anti-viral RNA silencing. The first tenuivirus RNA silencing suppressor identified is the p3 (NS3) protein from RHBV (NS5 for RGSV) ,163 Fig. RHBV p3 in rice . While pnfection . P3IP, anfection . Besidesnfection . A hypotnfection   protein, similar to RSV p3, functions as an RNA-silencing suppressor  Figure . RGSV p2 protein . Contrar protein . RSV p3  protein .Bunyavirales. However, they encode additional non-structural accessory proteins, and there are only a few reports suggesting functions in cell-to-cell movement or silencing suppression, yet the functions of most proteins are not determined experimentally. Until now, the majority of efforts by researchers have been at the epidemiological level and using next-generation sequencing technology to discover new species members of the genus. The recent development of an infectious clone for RRV creates the opportunity for reverse genetic studies to investigate the functions of various RRV proteins and to carry studies of virus–host interactions at the cellular and molecular levels [Emaraviruses encode the RdRp, N, Gn, and Gc proteins as for other plant- and animal-infecting members of the r levels .Some functional studies have identified RNA4 to encode the MP, and amino acid sequence alignments have allowed the extrapolation to suggest all emaravirus RNA4 segments encode the viral MP . The RLBSorbus intermedia encode p42.4 kDa and 27 kDa proteins, respectively, sharing significant homology with the FMV P4 MP [For many other species, RNA5 and RNA6 are suggested to encode virus MPs, but their origins seem to vary among species, and experimental investigations testing their functions are lacking . For exaMV P4 MP . The EurMV P4 MP . HPWMV RMV P4 MP ,109. As The HPWMV RNA7 and RNA8 encode the p7 and p8 silencing suppressor proteins, respectively  Figure . The p7 F. occidentalis has been used.The spread of orthotospoviruses relies on a complex of interactions between the virus, thrips vector, and host plant, which is additionally influenced by many (a)biotic factors. In many studies on this topic, and to which most studies described in this section relate to, the major species Bunyaviridae, e.g., Bunyamwera virus [Thrips are only able to transmit the virus when they feed on infected plants primarily during their larval L1 or L2 stages ,181,182.ra virus , pointinra virus ,187,188.ra virus . The atora virus .TSWV replicates in the midgut and then disseminates into the thrips body, where the virus next primarily localizes in the salivary glands as the (second) major site of replication ,182. ThrNeohydatothrips variabilis/Soybean vein necrosis orthotospovirus pathosystem, thrips fed on a combination of soluble peptides containing the “RGD” and the “R229” motifs, characteristic of cellular attachment domains and present in Gn of several orthotospoviruses from the American clade, reduced virus transmission by 67% [The viral Gn and Gc form the holo-spike complex enabling transmission of orthotospoviruses by thrips, and mutants hardly producing virus particles, but also accumulating defective-interfering (L-RNA derived) RNA molecules, are compromised in their transmission efficiency ,193. Feen by 67% .Although about 15 thrips species have been identified as vector for orthotospoviruses, they exhibit a vector competence towards certain orthotospovirus species only, and not all . While tF. fusca and F. tritici, a vector and non-vector of TSWV, respectively, has revealed some differences, but the relevance of those towards virus transmission still needs to be further investigated [Transmission of orthotospoviruses by thrips is (indirectly) affected by many other factors, often involving altered (preferred) feeding behavior on infected plants , and in which the thrips (indirectly) benefits from the virus, e.g., increased life span, fecundity, and offspring ,203,204.stigated . In somestigated ,214.Data also show that viral infection changes the plant metabolism and defense responses, turning these plants to be more conducive for thrips feeding/colonization ,203,214.F. occidentalis [Although Gn and Gc are indispensable for acquisition and transmission of orthotospoviruses by thrips, the NSs protein also appears needed for persistent infection and transmission. Studies on a collection of NSs-defective TSWV isolates showed that these could not be transmitted by dentalis . While tThe presence of thrips resistance in plants indirectly affects the acquisition and inoculation of the virus by thrips as well. While transmission from these plants is basically not affected, thrips show a lower fecundity and lower preference for these plants, and beneficial effects on virus transmission may thus be expected in outstanding crops . AlteredThrips tabaci have indicated the complexity of virus vector competency, in which the clonal type of T. tabaci (population (genetic) structure) and a specific interaction with the  virus isolate play a major role [F. occidentalis indicate the presence of hereditary traits involved in virus vector competence [Studies on jor role ,226,227.mpetence .Tenuiviruses are transmitted horizontally by delphacid planthoppers in a circulative-propagative manner, i.e., the virus multiplies in the insect vector. They are also vertically transmitted by viruliferous females to their offspring . ImmunofMost bunyavirids are enveloped viruses that enter arthropod cells through interaction between virus surface glycoprotein and host receptors, and in some cases, host receptor is identified as lectin or integrin ; on the Several viral proteins are involved in viral infection of the insect and horizontal transmission. The glycoprotein pc2 (NSvc2) is required for RSV entrance into the planthopper midgut cells. It acts as a helper component for transmission, the first described for a persistent propagative virus . In infeRecently, RSV p3 was found to interact with alpha-tubulin2 of the insect vector, mediating the passage of RSV through the midgut and salivary glands . MoreoveAs in plants, tenuivirus proteins interact with the host components involved in insect defense mechanisms. The angiotensin-converting enzyme of the small brown planthopper (SBPH) appears to play a role in the immune response against RSV transmission by planthoppers, although the mechanism is not well elucidated . RSV N bRSV reduces the fecundity of its vector by changing the expression of developmental genes in embryos . The preUnlike orthotospoviruses or tenuiviruses, there is little known about the transmission attributes of emaraviruses. This is because the vast majority of the more than 25 emaravirus species or tentative species have been discovered in the past decade and vectors have been identified for less than half of these species. Emaravirids are transmitted by eriophyoid mites, arthropods that are indiscernible to the naked eye as they average 0.2mm in length, and transmission studies are highly recalcitrant .Aceria cajani [Eriophyoids are largely monophagous, preferring to feed on one type of plant , which ma cajani ,249. Thea cajani . As knowa cajani . It is wa cajani ,253, sugPhytoptus pyri, a common pest of mountain ash, was tested positive for both the genomic and complimentary strands of EMARaV, a possible indicator of virus replication or part of the narrative that emaraviruses encapsidate both v and vc RNAs in their particle. Notwithstanding, a large amount of virus N protein was found to be present in the mite body, altogether leading to the hypothesis that emaraviruses replicate in their vector [The transmission mode of emaraviruses is largely unknown. Some emaraviruses are readily transmissible with short acquisition access periods of ≈15 min whereas others require significantly more time, sometimes several days . Once thr vector . In addiThere are recent excellent articles on the phylogeny of the Bunyavirales ,256, andThe last common ancestor of the Bunyavirales is most probably an invertebrate, possibly insect-infecting virus ,258, witThere are no studies that address how microevolution affects any of the aforementioned taxa, largely because reverse genetics platforms have only recently become available ,95,261. Orthotospoviruses, the better studied group of the cohort, have identical genome organization, composed of three RNAs and five proteins expressed using an ambisense strategy . Thrips N. benthamiana [Tenuiviruses have obvious genome plasticity, with members having genomes ranging from four to eight segments. Whereas the function of the core viral protein has been determined ,67,90, tthamiana , suggestthamiana .Emaraviruses are the most diverse of the group of the plant-infecting bunyavirids, given that their genome has between 5 and 10 segments and some proteins have no orthologs in the databases. This diversity has led to deliberation on the exact number of segments carried by each virus, and there are cases where emaravirus genomes have been revisited and expanded ,109. EmaOn the other hand, there are cases where one of the coding regions of the RNA variants has accumulated enough mutations that the duplicated genes become paralogs. PerMV presents an excellent example for both duplication and diversification. The N proteins encoded by the RNA3 variants are homologs sharing over 80% aa identities. The proteins encoded by the RNA6 variants have diversified to the point where these two share about 65% aa identity, whereas the third is much more diverse (<25% aa identity with the other two), presenting a possible paralog . The dupGroundnut ringspot and Tomato chlorotic spot orthotospoviruses [In addition to the genome plasticity, there is ample evidence that recombination and reassortment are important in the evolution of emaraviruses. In the case of blackberry leaf mottle-associated virus, there is evidence of both recombination and reassortment within the species . In PPSMoviruses .Phenuiviridae. Due to their unique features, i.e., these viruses have a bisegmented RNA genome of negative and ambisense polarity; encode for the RdRp, N, and MP (but not glycoproteins); and do not contain a viral membrane envelope, they have been classified into a newly proposed Coguvirus genus within the Phenuiviridae [Brassica [Rubodvirus [Although orthotospoviruses and tenuiviruses have already been known for many decades, and emaraviruses seemingly emerged only recently, the use of HTS/meta-genomics/transcriptomics to resolve plant viromes is boosting the discovery of new isolates/species belonging to the NSVs with segmented genomes. While tenuiviruses and emaraviruses were initially thought to be limited to monocots and perennial plants, respectively, recent HTS efforts have enabled the discovery of tenuiviruses and tenuivirus-like viruses (phenuivirids) in tulip, melon, a plant parasitic nematode, and a fungus ,271,272,iviridae ,274. AltBrassica  could bobodvirus . The  ,287,288.


To determine 1-year attributable healthcare costs of bronchiolitis.Using a population-based matched cohort and incidence-based cost analysis approach, we identified infants <12 months old diagnosed in an emergency department (ED) or hospitalized with bronchiolitis between April 1, 2003 and March 31, 2014. We propensity-score matched infants with and without bronchiolitis on sex, age, income quintile, rurality, co-morbidities, gestational weeks, small-for-gestational-age status and pre-index healthcare cost deciles. We calculated mean attributable 1-year costs using a generalized estimating equation model and stratified costs by age, sex, income quintile, rurality, co-morbidities and prematurity.We identified 58,375 infants with bronchiolitis . Total 1-year mean bronchiolitis-attributable costs were $4,313 per patient , with $2,847  spent on hospitalizations, $610 (95%CI: $594–627) on physician services, $562 (95%CI: $556–567)] on ED visits, $259 (95%CI: $222–297) on other healthcare costs and $35 ($27–42) on drugs. Attributable bronchiolitis costs were $2,765  vs $111 (95%CI: $102–121) in the initial 10 days post index date, $4,695  vs $910 (95%CI: $847–973) in the initial 180 days and $1,158  vs $639 (95%CI: $599–679) during days 181–360.Mean 1-year bronchiolitis costs were higher in infants <3 months old , those with co-morbidities  and with low birthweight .Compared to no bronchiolitis, bronchiolitis incurs five-time and two-time higher healthcare costs within the initial and subsequent six-months, respectively. Most expenses occur in the initial 10 days and relate to hospitalization. Bronchiolitis is a viral disease characterized by rhinorrhea, cough and respiratory distress, typically affecting infants under 12 months of age –10. NoneBronchiolitis carries high morbidity; it represents the leading cause of infant hospitalizations in the Western world, with increasing costs over the past several decades ,14,15. IPrevious assessments of bronchiolitis management have focused on relatively short-term costs, such as expenditures limited to hospitalizations ,21, impaTo address this knowledge gap, we conducted a cohort study of infants who presented to an ED or were admitted to a hospital in Ontario, Canada with acute bronchiolitis between April 1, 2003 and March 31, 2014, to determine the total 1-year bronchiolitis-attributable healthcare costs. We hypothesize that the overall healthcare costs among infants who experienced bronchiolitis in the first year of life, are significantly higher than the overall healthcare costs among infants who did not experience bronchiolitis, after matching on sociodemographic and clinical covariates.We conducted an incidence-based, propensity score matched cohort study to determine the total attributable healthcare costs in infants with bronchiolitis from the perspective of the healthcare payer .th Revision [ICD-10-CA] code J21 or J12.1) between April 1, 2003 and March 31, 2014 (exposed subjects). We assigned the first instance of ED visit/hospitalization for bronchiolitis as the index date. If a physician office visit for bronchiolitis occurred within two days prior to the ED visit/hospitalization, we used the date of the physician office visit as index date. We followed all study subjects for one year after the index date or until death. Residents of Ontario have access to universal public health insurance under the Ontario Health Insurance Plan (OHIP), the single payer for medically necessary services, such as hospital visits and medical procedures , and patients tended to live in lower-income neighborhoods in urban centers . Total mean costs attributable to bronchiolitis over a one-year period from index date were $4,313 per subject —Table 2. The greatest excess costs incurred were due to inpatient hospitalizations, followed by physician services, ED visits, other healthcare costs and drug costs. This trend was also observed for most of the initial 30-day, 180-day, and 360-day intervals from index date. Costs attributable to bronchiolitis were seven times higher during the initial 180 days compared to the latter 180 days post-index date (Table 2). Healthcare costs in infants with bronchiolitis compared to those without bronchiolitis were five times higher during days 0–180 and almost two-times higher during days 181–360 after index date (Table 2).Exposed infants who survived the first year of life had higher mean healthcare cost within the 360-day period following index date, both overall and across every 10-day interval within this period, compared to unexposed infants (Table 3). There were no significant differences in attributable 1-year costs incurred in males versus females, in infants living in rural versus urban areas or in those across neighborhood income quintiles (Table 3).Infants less than 3 months of age, those with at least one complex chronic condition and infants born prematurely had higher attributable costs over the 1-year period post-index date compared to their counterparts without these characteristics . The impact of potential misclassification is therefore expected to be small.While we accounted for baseline differences in known co-morbidities, other complex conditions which impact healthcare utilization and costs may not have been diagnosed prior to the index date. Therefore, we do not know whether such unidentified co-morbidities would be distributed equally among patients with and without bronchiolitis. However, we also included healthcare costs prior to the index date as a proxy for healthcare utilization due to co-morbidities and to account for healthcare-seeking behavioral differences of caregivers.Another limitation is that our study design allowed us to focus only on healthcare costs from a healthcare payer perspective . In Ontario, drugs are only publicly funded for (but not limited to) hospitalized patients and for outpatients aged ≥65 years or those on social assistance. Therefore, drug costs not publicly funded are not captured in our study. Further, indirect costs from the societal perspective  were not included. Therefore, our cost estimate is conservative.The strengths of our study include the availability of comprehensive, population-based data, which allowed us to include a broad set of variables in the propensity score, thus enabling us to account for patient-level differences between infants with and without bronchiolitis and to reduce bias. Further, our analysis accounted for all publicly funded healthcare costs and extended beyond the initial ED visit or hospitalization. Therefore, our study provides a comprehensive estimate of longer-term healthcare costs of bronchiolitis. Our findings are based on a large, province-wide, population-based bronchiolitis cohort managed over an 11-year period, with no socioeconomic, geographic or other restrictions, which increases the generalizability of the findings.In summary, we found that costs for infants with bronchiolitis were five-time and two-time higher compared to infants without bronchiolitis within the initial and subsequent six-month post-index date periods, respectively. The 10-day attributable costs were highest during the initial 10 days, and remained higher one year later. The majority of the bronchiolitis-attributable costs relate to hospitalizations.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question “what is a virus?”.The discovery of  Acanthamoeba polyphaga Mimivirus  . ORF132 ltimeric  suggestiChlorella viruses, and have been linked to K+ protein channels [Chlorella virus-host interactions. This phenomenon, lowering the energy barrier, allows the viral DNA release into the host. However, it appears to differ from those observed in pandoraviruses. Indeed, pandoraviruses seem to harbor predicted genes with low similarity with all enzymes of the cellular TCA cycle and most importantly, a functional isocitrate dehydrogenase, a key enzyme of this cycle. Moreover, the membrane potential of P. massiliensis virions is affected by acetyl-CoA, a well-known regulator of the TCA cycle. One may assume that a complete TCA cycle can occur if all the enzymatic components are present in a system. Herein, our findings suggest the possible existence of an autonomous, at least partial, TCA pathway potentially involved in energy production, or in another, unknown metabolic pathway, in a virus. As the TCA cycle plays a key role in anabolism and catabolism of many biomolecules, it also can also be hypothesized that these viral genes may be involved in redirecting amoeba carbon metabolism.Experimental evidence of a virus encoding enzymes of a partial TCA cycle opens a new horizon in giant virus research. For each experiment, critical negative controls were carried out to avoid false positive results. Membrane potential and in particular, membrane depolarization, have been previously described in channels , 81. TheIt is known that viruses are major drivers in nutrient and energy recycling within natural environments and in the ecology and evolution of their cellular hosts –85. In tP. massiliensis undermines the last known historical viral hallmark, the lack of the Lipman system. Thus, our findings presented herein raise critical questions concerning whether pandoraviruses can still be accurately classified biologically as viruses and renew deep-rooted arguments regarding the living nature of viruses in general.In conclusion, Supplementary file 1Supplementary file 2Supplementary file 3Supplementary file 4Supplementary file 5Supplementary file 6Supplementary figure 1Supplementary figure 2Supplementary figure 3Supplementary figure 4Supplementary figure 5Supplementary figure 6Supplementary figure 7Supplementary figure 8Legends for supplementary files/figs


Previous literature has examined the societal costs of mental illness, but few studies have estimated the costs associated with mental well-being. In this study, a prospective analysis was conducted on Danish data to determine 1) the association between mental well-being (measured in 2016) and government expenditure in 2017, specifially healthcare costs and sickness benefit transfers.Data stem from a Danish population-based survey of 3,508 adults (aged 16 + years) in 2016, which was linked to Danish registry data. A validated scale (WEMWBS) was used for the assessment of mental well-being. Costs are expressed in USD PPP. A two-part model was applied to predict costs in 2017, adjusting for sociodemographics, health status , as well as costs in the previous year (2016).Each point increase in mental well-being (measured in 2016) was associated with lower healthcare costs  and lower costs in terms of sickness benefit transfers  per person in 2017.Estimated reductions in costs related to mental well-being add to what is already known about potential savings related to the prevention of mental illness. It does so by illustrating the savings that could be made by moving from lower to higher levels of mental well-being both within and beyond the clinical range. Our estimates pertain to costs associated with those health-related outcomes that were included in the study, but excluding other social and economic outcomes and benefits. They cover immediate cost estimates  and not those that could follow improved mental well-being over the longer term. They may therefore be considered conservative from a societal perspective. Population approaches to mental health promotion are necessary, not only to potentiate disease prevention strategies, but also to reduce costs related to lower levels of mental well-being in the non-mental illness population. Our results suggest that useful reductions in both health care resource use and costs, as well as in costs due to sick leave from the workplace, could be achieved from investment in mental well-being promotion within a year. The costs of mental health problems to society are substantial. In Europe, costs have been estimated to be more than 4% of its GDP—or over €600/$680 billion—across the 28 EU countries , with 1.This study builds on a concept of mental well-being implicit in the Warwick–Edinburgh Mental Well-being Scale (WEMWBS), covering both hedonic (feeling good) and eudaimonic (functioning well) aspects, with both aspects being integral parts of the overall construct . FunctioWe identified seven studies that explored the predictive value of well-being measures specifically in regards to healthcare utilization/expenditure and loss to productivity –18. All Studies which add to the evidence base of costs associated with different levels of mental well-being contribute to the ability of governments to secure financial sustainability. This is particularly pertinent as many countries and regions face budget constraints and may be inclined to cut funding for initiatives and interventions devoted to mental health promotion. Lack of empirical evidence on the economic benefits of mental well-being may explain why policy makers incline towards cutting funding for such initiatives. In this study, using a large sample of the adult Danish population, we assessed the association between mental well-being and government expenditure in Denmark, specifically healthcare costs and sickness benefit transfers. Based on the aforementioned evidence, we hypothesized that higher mental well-being scores  would be associated with lower healthcare costs and sickness benefit transfers in the following year.Our data came from The Danish Mental Health and Well-Being Survey 2016 (DMHWBS2016) , which iAll costs were extracted from Statistics Denmark for the year 2016 and 2017. This cost analysis utilized data from Danish national registers using each respondent’s anonymized civil registration number linked to the DMHWBS2016. Costs comprised (1) healthcare costs , and (2) costs in terms of sickness benefit transfers . Unit costs for general practitioners and specialists are based on the current national health insurance rate . ChargesAll healthcare costs were aggregated for:  the fullFor a detailed description of cost components, see Appendix 1. All costs used for analysis are in 2016/2017 prices (DKK) and results were subsequently converted to international dollars  using an online conversion tool  .The Warwick–Edinburgh Mental Well-being Scale (WEMWBS) is a validated measure used to monitor mental well-being in the general population . The scaAll sociodemographic variables were extracted from Statistics Denmark for the year 2016. The sociodemographic variables were: age; sex ; migration background ; marital status ; education ; employment status ; and income . Since 6.8% of data on income were missing, a ‘missing’ category was created for this variable.Three variables pertaining to health status and health behaviors were included. The first two came from the register. To classify the presence of chronic conditions, we used the Charlson Comorbidity Index (CCI). It is based on 19 different medical conditions , each weighted and assigned 1–6 points according to its potential impact on mortality, derived from relative risk estimates . The CCISTATA version 14 was used to perform all analyses. Following recommendations regarding the use of healthcare cost data characterized by heavily tailed and truncated distributions, we applied a two-part model , 29. A tThus, the two-part model allows for a separate investigation of the effects of a predictor on the extensive margin  and on the intensive margin . Subsequently, the incremental effects of the predictor on the outcome for the combined probit and GLM are calculated .N = 1839).The statistical analyses conducted were as follows. First, simple unadjusted graphs (fitted lines) were drawn to illustrate the relationship between the continuous WEMWBS score in 2016 and costs in 2017. Subsequently, the associations between the continuous WEMWBS score in 2016 and costs in 2017 were estimated adjusting for covariates. For each analysis estimating costs, we conducted a model 1 that adjusted for age, sex, migration background, marital status, education, employment status, income, and 2016 costs, and a model 2 that adjusted for all the aforementioned variables as well as chronic conditions, the number of psychiatric conditions, and physical activity. Both these two-part models were performed using the continuous WEMWBS scale as the predictor. In analyses where the outcome was sickness benefit transfers, we did not adjust for employment status in either model 1 or model 2, since sickness benefit transfers imply absence from employment or education. Also, since this outcome only pertains to individuals that were or would otherwise have been active on the labor market, the sample was restricted only to the working-age population, i.e., 16–64 years old . Table In this study, we set out to estimate the extent to which continuous mental well-being scores in one year predicted healthcare costs/sickness benefit transfers in the subsequent year. Higher mental well-being  in 2016 was associated with lower healthcare costs and sickness benefit transfers in 2017. That is, after adjustment for a wide range of covariates including mental/chronic illnesses, physical activity, and costs in the previous year, each point increase in mental well-being was associated with lower healthcare costs  and lower costs in terms of sickness benefit transfers  per person in 2017.In line with previous findings –18, our Our results also suggest that improvements in mental well-being could generate a positive return on investment in the very short term. An intervention costing $65.6  per individual and generating a one-point increase in the WEMWBS scale would - by lowering the need for health care services and sick leave over the course of a year - reduce expenditure and free up scarce resources equivalent to the amount invested. Such assertions depend on evidence to show that mental well-being can be improved over a short time period, and the evidence base for this is now growing , 33–35. Denmark saw a dramatic 46% increase in public healthcare expenditure from 2000 to 2017 , with 16Mental well-being is modifiable, and supporting efforts to foster mental well-being throughout populations should be an end-goal in policy and practice . Policy Elsewhere, countries such as New Zealand have recently placed an emphasis on the promotion of well-being, particularly for younger people, in the country’s first-ever well-being budget . ScotlanUniversal mental health promotion implies fostering behaviors and environments known to protect and enhance mental well-being. Such initiatives may encourage individuals to engage in mentally healthy behaviors, such as those that enhance awareness and self-regulation , keeping active in various ways, maintaining contact with social ties and participating socially, as well as getting involved in meaningful challenges and causes. Initiatives may also support community organizations and municipalities in fostering environments that provide opportunities to engage in mentally healthy behaviors . ImportaThe latter point also applies in terms of healthcare utilization, as research has already demonstrated that higher community well-being (counties or zip codes with higher average well-being) is associated with reduced healthcare expenditure within those communities , 18. MetSome strengths and limitations should be kept in mind when interpreting the results. Major strengths include the prospective design, the use of a validated scale for measuring mental well-being, and the use of a population-based survey linked with national registers. This approach made it possible to make direct links between mental well-being in one year and cost outcomes expressed in monetary terms in the subsequent year, as well as a range of register-based covariates. Also, since the outcomes and most covariates were from the register rather than from the survey, common methods as a result of single-source self-reported data are not an issue in this study. Some limitations are as follows: The response rate was 34%, and while this is not unusual for web-based surveys, selection bias cannot be ruled out. Response rates were higher for women, individuals aged 45 years old and above, individuals with higher education (tertiary), individuals who were married or in a registered partnership, employed individuals, individuals with a Danish (non-immigrant) background, and individuals with a higher income . We haveIn this study, our final results are based on analytical models where we adjusted for health status and health behavior (apart from demographics and socioeconomic factors). We did this to minimize confounding of health factors, but there is a possibility of overadjustment, since our models already took into account past healthcare utilization. It is also important to consider that healthcare costs and transfers are highly correlated from one year to the next due to ongoing illness. Some of these costs may respond positively to improvements in mental well-being, for example through improvements in self-care, health-related lifestyles, and greater resilience to stress. Our approach is conservative as it excludes all longer-term ongoing positive impacts on chronic illness, and instead focuses solely on the costs which are most immediately associated with mental well-being.Some additional reflections should also be made in terms of the scope of our study and its implications. In this study, we were not able to estimate the potential savings of various other related cost outcomes due to lack of data, for example outcomes pertaining to presenteeism, short-term sickness benefit transfers or municipal care services. Also, our study design did not allow for estimating the costs of disability pensions since well-being does not involve a diagnosis of which disability pensions are based, but other studies have documented inverse associations between well-being and intentions to retire early  as well The results of the present study support and expand prior findings in that population mental well-being predicts future expenditure (expressed in USD PPP) pertaining to healthcare costs and sickness benefit transfers. Higher mental well-being  in 2016 was associated with lower costs in 2017, i.e., each point increase in mental well-being was associated with lower healthcare costs  and lower costs in terms of sickness benefit transfers  per person in 2017. The relationship was linear, implying that moving from low to high mental well-being across the entire continuum is associated with considerable cost savings. The results were robust when considering differences in sociodemographics, psychiatric and somatic health status, as well as health behavior. Estimates cover the cost outcomes included in this study and pertain to the short-term reductions in costs associated with increases in mental well-being. Our estimates must therefore be considered conservative from a societal perspective. Our results suggest that investing in the promotion of population mental well-being, while being desirable in its own right, would have the additional benefit of curbing a wide range of costs. Universal mental health promotion initiatives that focus on moving all segments of the population towards higher levels of mental well-being could free up resources and reduce costs in the short term, potentially being cost neutral, as well as generate cost savings for society in the longer term.


Vaccinium spp.) plants are exposed to existing and emerging viruses as a result of expanding acreage of blueberry plantations across the world, primarily in North America. Since blueberry is cultivated in areas where there are wild Vaccinium spp., there is increasing risk of virus movement between wild and cultivated blueberries. This is theoretically possible because viruses can spread from commercial cultivars to native species and vice versa causing the spread of existing and new viruses. The occurrence of these viruses in blueberry can be devastating to the industry considering the cost for cultivation and production of this perennial crop. However, the advent of high-throughput sequencing and bioinformatic sequence analysis have allowed for rapid identification of known and novel viruses in any crop including blueberry, thus facilitating proper intervention in response to serious viral diseases. In this paper, we aim to focus on the current status of known and novel viruses emerging in blueberry worldwide, which may impact the blueberry industry.Blueberry ( Vaccinium, in the Ericaceae family, is organized into sections comprised of important fruit crops including blueberries, which are mostly derived from the section Cyanococcus [The genus nococcus . Bluebernococcus . Other mnococcus .V. corymbosum L.), lowbush (V. angustifolium Ait), and rabbiteye (V. virgatum Aiton) [V. corymbosum). The southern United States  predominantly grow southern highbush blueberry  [The three species of blueberries in the section Cyanococcus produced commercially include highbush (m Aiton) ,5. Highbm Aiton) ,7. The nii Camp) ,8.Vaccinium spp. are exposed to existing and emerging viruses as a result of expanding acreage of blueberry plantation across the world, primarily in North America [Vaccinium spp., there is increasing risk of virus movement between wild and cultivated blueberries. This is theoretically possible because viruses can move from commercial cultivars to native species as well as from wild species to cultivated blueberries, causing the spread of existing and new viruses. The cost for cultivation and production of a perennial crop such as blueberry is significant, hence viral diseases in this crop can be economically devastating [ America ,10. Sincastating . Viral dIlarvirus belongs to the family Bromoviridae, along with six other genera. Woody plants are the major type of plants that are infected by ilarviruses, which are spread mechanically by thrips feeding on virus-infected pollen grains or by transporting virus-infected pollen grains [The genus n grains .Blueberry shock virus (BlShV) has been found in California, New York, Oregon, Pennsylvania and Washington, in the US, and Nova Scotia and British Columbia in Canada. During its first appearance in Washington in 1980, blueberry shock virus (BlShV) symptoms on blueberry were initially confused with blueberry scorch virus (BlScV) symptoms . AlthougIlarvirus, according to a serological test using indirect ELISA. BlShV is classified as an ilarvirus based on its physicochemical properties.BlShV virions are nonenveloped, quasispherical, and have a diameter of about 26–29 nm. They are made up of 180 CP subunits, each with a MW of about 27 kDa . BlShV hLike other ilarviruses, pollen is the main mode of transmission for BlShV, with a low level of transmission by seed . The priClosteroviridae contain four genera while other species are yet to be assigned into a genus. Viruses in this family are commonly vectored by in a semipersistent manner by aphids, whiteflies, pseudococcid mealybugs or soft scale insects. Seed transmission is unknown, and experimental transmission via mechanical injection is difficult or impossible [The family possible .Closterovirus genus, but they differ by more than 10%. Since BVA failed to form a clade with other closteroviruses based on phylogenetic analysis of the RdRp, HSP70h, and CP, it was proposed as a new species. Transmission of BVA has not been effective in a study using mechanical and aphid vector, thus its transmission mode is not yet known. Since BVA has not been associated with specific symptoms in single infections, the extent of economic damage to the blueberry industry is yet to be determined.Blueberry virus A (BVA) was isolated for the first time from a highbush blueberry cultivar called ‘Spartan’ in Japan, and later was reported in Canada and the US ,14. BlueVaccinium spp. belong to the genus Nepovirus from the Secoviridae family  and Pro-Pol regions, respectively [The most common viruses that infect e family . Nepovirectively . The majChenopodium quinoa Willd. quinoa, Luffa cylindrical (L) Roem, and Nicotiana benthamiana produced chlorotic spots in systemic leaves following mechanical inoculation of BlSV, suggesting that the virus is associated with latent infection of blueberry [Blueberry latent spherical virus (BlSV) was the first nepovirus isolated from asymptomatic highbush blueberry in Japan . Graft ilueberry .The virus belongs to subgroup C of nepoviruses based on the genome organization. It shares closest amino acid sequence similarities to the RdRp of peach rosette mosaic virus (57%) and CP of apricot latent ringspot virus (43%) . The traSymptoms of mottling and distortion on the leaves of highbush blueberries associated with blueberry leaf mottle virus (BLMoV) were first observed in 1977 in Michigan (USA) . The virNepovirus genus, it is spread randomly by honeybees through an infected pollen and possibly by seeds [Vaccinium spp. bushes surrounding the field, implying virus movement between cultivated and wild areas [Although BLMoV is a member of nematode transmitted by seeds ,18. BLMoby seeds . BLMoV wld areas . BLMoV cld areas .Vaccinium darrowii cv. Jubilee 83) in New Zealand in 2013, along with other isolates from various hosts [Prunus avium L.) in England in 1955, and was later discovered in other European countries, North America, and other regions of the world, infecting a number of herbaceous and woody plants, including fruit trees of valuable horticultural crops [Rubus sp. but the characteristic symptoms in blueberry were not known [The first occurrence of cherry leaf roll virus (CLRV) in blueberry was only reported recently in a southern highbush blueberry species  which are conserved between the 8 kb RNA1 and 7 kb RNA2 genomes, a feature seen in cherry and rhubarb isolates as well. Based on phylogenetic analysis of the CP and Pro-Pol regions, CLRV was confirmed to be closely related to other members of nepovirus-subgroup C ,55. UnliPeach rosette mosaic virus (PRMV) was first identified as a causal agent for rosette mosaic of peaches in the 1970′s, before being reported in grape and later in highbush blueberry ,69,70. TNepovirus genus [Xiphinema americanum Cobband and Longidorus diadecturus Eveleigh and Allen, and is classified as a soilborne virus [Chenopodium quinoa provides a more effective detection than ELISA in some circumstances [A complete genome sequence of PRMV confirmed its status in subgroup C of us genus ,71. PRMVne virus . Serologmstances . HoweverA necrotic ringspot disease associated with tobacco ringspot virus (TRSV) in blueberry was first identified in New Jersey in the early 1960s ,73. The X. americanum, which is also seed transmitted in some other weeds and crops [TRSV is classified as a distinct member of Subgroup A nepovirus based on its serological relationship and the existence of RNA2 in both the M and B components of virus particles. TRSV is transmitted in blueberries by the nematode nd crops . Plant snd crops .Tomato ringspot virus (ToRSV) was first discovered in blueberries in 1972 , but it was only found in highbush varieties grown in the US , Canada, and Chile ,74,75,76Nepovirus based on the almost inseparable middle and bottom components of its nucleoprotein, as well as the presence of high molecular weight RNA2 [Xiphinema spp. nematodes and is readily transmissible to a variety of herbaceous species by sap inoculation [ToRSV, like other blueberry-infecting nepoviruses, belongs to subgroup C of genus ght RNA2 . Completght RNA2 ,79. ToRSculation ,78,80. Sculation . Cautionculation .Secoviridae contains five genera while other species are yet to be assigned into genus. The majority of known members are significant plant pathogens mostly infecting dicotyledonous plants. Most secoviruses are transmitted by known biological vector and can be spread easily via seed or pollen [The family r pollen .V. darrowii, a new host of SLRSV [Strawberry latent ringspot virus (SLRSV) had not been found in blueberry until recently in New Zealand in 2014, where it was discovered in of SLRSV . SLRSV wof SLRSV , and hasof SLRSV ,83. Sympof SLRSV .Nepovirus and Sadwavirus, SLRSV is currently placed in an unassigned genus of the family Secoviridae [X. diversicaudatum and X. coxi, and is seedborne in some plant species such as raspberry and celery [Phylogenetic analysis of the CP region showed that the three SLRSV blueberry isolates from New Zealand, including one strawberry isolate from the United States, form a cluster with those from North America. This indicates that the virus was introduced to New Zealand by a single event. After being formerly assigned in the genus oviridae ,84,85. Toviridae . Howeverd celery .Sobemovirus belongs to the family Solemoviridae, along with two other genera. Each virus species has a limited natural host range, which includes monocotyledonous and dicotyledonous plant species. Sobemoviruses are spread primarily through mechanical injury of host plants and can be transmitted by a variety of insect species [The genus  species .V. corymbosum), with infected bushes losing up to 25% of their yield [Blueberry shoestring virus (BSSV), a sobemovirus, was first identified as the cause of shoestring disease in blueberry in New Jersey ,31, and ir yield . Shoestrir yield .Illinoia pepperi) vectored the transmission of BSSV into blueberry plants in a persistent, circulative manner [The BSSV virion is a nonenveloped isometric particle with a diameter of 28 nm. It has a single-stranded RNA genome with four putative ORFs . The 15.4 kDa movement protein, a 65.55 kDa polyprotein protein (Protease-VPg), a 62.22 kDa RNA-dependent RNA polymerase protein, and a 27.85 kDa coat protein were encoded by ORF1 (nt No. 85-492), ORF2a (nt No. 462-2204), ORF2b (nt No. 1796-3400), and ORF3 (nt No. 3186-4001), respectively . BSSV ise manner . AlthougBlunervirus has been recently classified in the family Kitaviridae.The genus V. virgatum) have not been infected with BNRBV, while southern highbush cultivars are known to be susceptible. Infected blueberries had distinct necrotic rings with green cores, but when the rings fused, they resembled the symptoms of fungal diseases (Blueberry necrotic ring blotch virus (BNRBV) was first discovered in southern highbush blueberries showing blueberry necrotic ring blotch symptoms in Georgia (USA) in 2006 . Since tdiseases . Early dCilevirus and Higrevirus, but it was recently assigned to a new genus, Blunervirus [The BNRBV genome is approximately 14 Kb long and consists of four RNA segments with seven ORFs  ,30. RNA1nervirus ,91.Based on its protein sequence similarity to citrus leprosis virus, BNRBV is most likely transmitted by an eriophyid mite. Transmission studies of BNRBV using eriophyid mite are currently being performed in Florida and Georgia to test this hypothesis ,93. BNRBOphiovirus is currently the only genus recognized under the family Aspiviridae (formerly Ophioviridae). Trees, shrubs, vegetables, and bulbous or corm-forming ornamentals, including monocots and dicots, are natural hosts for aspiviruses. Four of the seven species in the family are known to be soil-transmitted [nsmitted .Although mosaic disease of blueberry was initially thought to be a physiological disorder, it was discovered to be related to viruses in the 1950s due to its graft-transmissibility . After iV. pallidum), until it was recently reported in wild highbush, V. corymbosum in Florida [The causative agent associated with blueberry mosaic disease has yet to be confirmed due to the difficulties in characterizing the agent. However, blueberry mosaic associated virus (BlMaV) has been discovered in mosaic-affected blueberries as well as asymptomatic plants in North America. It has been proposed as a possible cause of blueberry mosaic disease . BlMaV h Florida ,97,99.Ophioviridae, the Ophiovirus, based on phylogenetic analysis of the RdRp region. Based on phylogeny clustering and genome arrangement, BlMaV is closely related to citrus psorosis virus (CPsV) [BlMaV is proposed as a new member of the only genus in the family s (CPsV) . The naks (CPsV) . The genSimilar to its closest relative, CPsV, the natural vector of BlMaV is still unknown although other ophioviruses are transmitted via fungal spores, suggesting that the BlMaV could have a similar soilborne vector . The praCarlavirus belongs to the family Betaflexiviridae. Some carlaviruses can infect a broad variety of experimental hosts, although some have limited natural host ranges. The majority of species are nonpersistently transmitted by aphids and are mechanically transmissible.Blueberry scorch and Sheep Pen Hill diseases were discovered on highbush blueberries in the states of Washington and New Jersey in the USA in the 1980s. Different strains of the same BlScV virus, a carlavirus, were identified as the causal agent ,105,106.Alphaflexiviridae, along with other members of the genus Carlavirus, based on the organization and sequence of the viral genome as well as serological relationships [BlScV virions are nonenveloped, flexuous particles (690 nm long × 14 nm wide) made up of a 33,500 kDa capsid protein encasing an 8.5 kb positive-sense ssRNA ,106  with green mosaic symptoms collected from Burlington County, New Jersey . The newBGMaV has a vitivirus-like genome structure, containing a ~7.5-kbp ssRNA molecule plus sense with five ORFs and a polyadenylated 3′ terminus. BGMaV’s ORF1 encodes a putative viral replicase (196K); ORF2 expresses a 16-kDa protein with no known function. Movement protein, CP, and nucleic acid binding protein are encoded by ORFs 3–5, respectively. BGMaV was inferred as a novel vitivirus based on phylogenetic analyses of the CP and conserved motifs of RdRp . The discovery of BGMaV necessitates its inclusion in blueberry certification programs in order to ensure consistent propagation and clean source of plant stocks.Betaflexiviridae, containing only two approved species (Potato virus T and Prunus virus T). The viruses in this genus has no recognized vector yet.The tepoviruses are members in the family A novel tepovirus, tentatively named as blueberry virus T (BlVT), has been recently discovered through metagenomics approach in blueberry cultivar ‘Gulf Coast’ and ‘Windsor’ from Island Grove, Florida . The speTepovirus. Similar to BGMaV, the discovery of BlVT has prompted the need to incorporate this virus in the blueberry certification program for the production of BlVT-free blueberry plants.BlVT genome is similar to those of tepoviruses, consisting of three overlapping ORFs encoding for RdRp (5457 nt), MP (1146 nt), and CP (663 nt), which makes up the 7.2-kbp genome. Phylogenetic analysis of the putative RdRp and CP encoded by BlVT suggested that this virus belongs to the genus Amalgavirus is the only genus containing plant viruses belonging to the family Amalgaviridae, a recently described group of dsRNA viruses. There were only four approved species in this genus, including blueberry latent virus (BlLV).The genus When a new disease known as blueberry fruit drop disease was detected in the Pacific Northwest  in the US in the early 2000s, blueberry latent virus (BlLV) was unwittingly discovered ,42. BlLVPartitiviridae family [Amalgavirus in the Amalgaviridae family, with STV as the type species [Despite the fact that blueberry fruit drop was later discovered to be unrelated to BlLV, the virus was studied further, resulted in the isolation of a 3.5 kb dsRNA molecule belonging to a virus now known as BlLV . BlLV’s e family ,115. As  species . It was  species ,42,117.BlLV is transmitted efficiently by seeds although in the absence of movement protein, implying that it replicates in its host during cell division. The lack of symptoms in certain BlLV-infected highbush cultivars has led to the conclusion that the presence of these viruses in blueberries is not a cause for concern yet.Soymovirus belongs to the family Caulimoviridae, with four approved species reported so far. The host range of soymoviruses are limited to one or two plant families and their vectors are still not known.The genus Red ringspot disease originally was described in New Jersey with associated symptoms observed on highbush blueberry in the 1950s  and sincSoymovirus in the family Caulimoviridae [Soymovirus have a genome that encodes for eight proteins with discontinuities in both the transcribed and nontranscribed strand. These gaps are sealed upon infection of the virus into the host cell. The closed dsDNA is then transcribed into mRNA in the nucleus by host DNA-dependent RNA polymerase. This serves as a template for synthesis of viral proteins and reverse transcribed into new copies of dsDNA genomes. New virions are released following encapsidation of the new dsDNA genomes [BRRV, the causal agent for red ringspot disease in blueberry, is a paratretrovirus that belongs to the genus oviridae ,119. BRRoviridae ,120. Mem genomes .Reliable diagnostic tests are available for BRRV and commonly involve conventional polymerase chain reaction (PCR). BRRV specific primers from New Jersey are used since no reliable detection is available for a routine enzyme linked immunosorbent assay (ELISA) test , and theSoymovirus genus remain unknown, though the red ringspot disease can be transmitted through grafting and softwood cuttings [To date the vector for BRRV and other members of the cuttings ,94. Infecuttings . Althougcuttings . The unscuttings .North America used to be the center for blueberry production, accounting for over 80% of global production from 2008–2010 . The proBetaflexiviridae, in within the last 3 years. Interestingly, the metagenomics study which has uncovered the new tepovirus have also led to the discovery of BlLV and BlMaV for the first time in Florida, and the first to demonstrate the occurrence of BlMaV in wild highbush [A comprehensive review of blueberry and cranberry viruses was published almost a decade ago . Althoughighbush .Blueberry producers should be aware of the viruses present in their respective area and develop a proper control strategy to prevent an outbreak. In North America, a total of nine viruses  have been reported as high risk due to their frequent occurrence and widespread in different production regions . In cont


Due to the failure of virus isolation of the Omicron variant in Vero CCL-81 from the clinical specimens of COVID-19 cases, an initial in vivo and subsequent in vitro approach was utilized for the isolation of the virus. A total of 74 oropharyngeal/nasopharyngeal specimens were collected from SARS-CoV-2 positive international travellers and a contact case at Delhi and Mumbai, India. All the specimens were sequenced using next-generation sequencing and simultaneously inoculated onto Vero CCL-81 cells for virus isolation. Subsequently, two omicron positive specimens were inoculated into Syrian hamsters for two passages. The initial passage of the positive hamster specimens was inoculated onto Vero CCL-81 cells. The clinical specimens, hamster specimens, and Vero CCL-81 passages were sequenced to assess the mutational changes in different host species. The replication of the Omicron variant in hamsters was confirmed with the presence of a high viral load in nasal turbinate and lung specimens of both passages. The successful isolation of the virus from hamster specimens with Vero CCL-81 was observed with cytopathic effect in infected cells and high viral load in the cell suspension. The genome analysis revealed the presence of L212C mutation, Tyrosine 69 deletion, and C25000T nucleotide change in spike gene of hamster passage sequences and an absence of V17I mutation in E gene in hamster passage sequences, unlike human clinical specimen and Vero CCL-81 passages. No change was observed in the furin cleavage site in any of the specimen sequences, suggesting intact pathogenicity of the virus isolate. Our data demonstrated successful isolation of the Omicron variant with the in vivo method first followed by in vitro method. The virus isolate could be used in the future to explore different aspects of the Omicron variant. The ability of SARS-CoV-2 to rapidly mutate has been the biggest challenge the world has faced while responding to the COVID-19 pandemic. Speculation and the anticipation of SARS-CoV-2 becoming endemic have come to an end with the recent emergence of a heavily mutated variant, Omicron (B.1.1.529). The fifth variant of concern (VOC) was first reported by South Africa followed by Botswana and Hong Kong in November 2021 .The global scientific community is investing enormous effort to determine the susceptibility, transmissibility, immune escape, and severity associated with Omicron. Such an emergency demands the availability of Omicron virus isolate. Many variants of SARS-CoV-2 have been isolated successfully utilizing Vero, Huh7, and human airway epithelial cells. Various cell lines such as Vero CCL-81, Vero SLAM, MA104, BGM, and Caco-2 have also supported the replication of SARS-CoV-2 ,6. Recenn = 73) and contact case (n = 1) from Delhi and Mumbai, India were collected during 10 October to 13 December 2021. These specimens were transported under cold chain to ICMR-National Institute of Virology, Pune, India.The oropharyngeal/nasopharyngeal swabs of SARS-CoV-2 positive  international travellers (2 incubator at 37 °C and observed daily for cytopathic effect (CPE) under an inverted microscope . Cellular morphological changes were recorded using a camera . Cultures that showed CPE on PID-5 were centrifuged at 4815× g for 10 min at 4 °C; the supernatants were processed immediately or stored at −86 °C. Further, those that showed CPE were grown in T-25 flasks at P-2 and titration was performed after serial dilution. Tissue culture infective dose 50 per cent (TCID50) values were calculated by the Reed and Muench method. The cells were examined microscopically for cellular morphological changes following inoculation  characterized by whole genome sequencing were selected for in vivo virus isolation. The Syrian hamsters (8–10 weeks old) were anesthetized using the Isoflurane. A hundred microlitre volumes of each specimen were inoculated through the intranasal route into two hamsters. The nasal turbinate (NT), lung specimens of passage 1 (P-1) were further passaged into new batch of hamsters (P-2). The hamsters were sacrificed and NT, lung specimens were collected on the 3rd day post-infection (DPI). NT and lung tissues (20% suspension) homogenates were prepared in sterile Minimum Essential Medium  using a homogenizer and were screened using rRT-PCR [https://www.hiv.lanl.gov/cgi-bin/HIGHLIGHT/highlighter.cgi, accessed on 28 December 2021) <0.05 were considered to be statistically significant.The clinical specimens, hamster specimens, and virus isolates were sequenced using next-generation sequencing with the Illumina MiniSeq Machine. Briefly, the ribosomal RNA depletion was performed using Nebnext rRNA depletion kit (human/mouse/rat) followed by cDNA synthesis using the first strand and second synthesis kit. The RNA libraries were prepared using TruSeq Stranded Total RNA library preparation kit. The amplified RNA libraries were quantified, normalized and loaded on the Illumina sequencing platform . Referenn = 2), B.1.617.2.106 (n = 1), B.1.617.2.120 (n = 1), B.1.617.2.122 (n = 2), B.1.617.2.125 (n = 3), B.1.617.2.4 (n = 2), B.1.617.2.43 (n = 1), B.1.617.2.6 (n = 2), B.1.617.2.85 (n = 2), AY.122 (n = 11), AY.125 (n = 4), AY.36 (n = 1), AY.4 (n = 4), AY.4.2.1 (n = 1), AY.43 (n = 4), AY.46 (n = 1), AY.5.4 (n = 1), AY.89 (n = 1), AY.9.2 (n = 2)) while 15 were identified as B.1.1.529. Among the 15 Omicron cases , 14 were international travellers from United Arab Emirates, South Africa, Nigeria, Tanzania, France, UK, Germany, The Netherlands, and one contact case from Mumbai. Thirteen were asymptomatic while two had mild cold, cough, and body ache.The complete genome (>98%) were retrieved from 61 clinical specimens. Forty six sequences belonged to Delta derivatives (Delta (n = 4), AY.122 (n = 2), AY.89 (n = 1), AY.46 (n = 1), AY.4 (n = 1), AY.4.2.1 (n = 1)). The Omicron positive clinical specimens were blindly passaged thrice; however, replicating virus could not be detected using rRT-PCR and no CPE was observed [An attempt of isolation using Vero CCL-81 yielded 10 Delta derivatives  demonstrated gRNA copies of 2.6 × 1010 and 6.6 × 1010 in NT and lungs, respectively, on 3rd DPI  and lung specimen of one of the hamster   inoculated onto Vero CCL-81 cells (P-1) showed evidence of cell rounding and detachment from post infection day (PID)-4. Syncytial cells formed large cell masses that increased in size and number as the infection progressed. Enhanced CPE was noted in P-2 at PID-3. Incomplete cell detachment from the tissue culture plate surfaces, rounding and refractive appearance of infected cells was observed A. The ha8 × 1010 B. The daThe molecular characterization of the virus isolates obtained with in vivo and in vitro method confirmed them as Omicron A. The OmA gain of an SNV expand was observed at the E gene (G26293A (V17I)) of P-1 Vero CCL-81 isolate, while it was absent in clinical as well as hamster sequences. Further, an SNV was also observed at M gene (C26577G (Q19E)) of hamster P-1 which was not observed in the clinical sample, Vero CCL-81 P-1, or hamster P-2. An in-depth analysis would be required to understand the gain or loss of this specific mutation.The frequency of the N440K amino acid change was observed in the hamster sequences (100%) and in the human clinical sequences (68.5%). The Vero CCL-81 passage demonstrated the decrease by 86.0% in Vero CCL-81 P-1 of the hamster (P-1) and 71.79% in Vero CCL-81 (P-1) of the hamster (P-2). The variation in the frequency of the N440K in human clinical specimens, hamster specimens, and Vero CCL-81 passages could be due to the adaptive mutations in different host species. A recent study linked this mutation to be associated with higher infectious titre . FurtherThis study reports the isolation of the Omicron variant using the in vivo followed by in vitro method of virus culture. Until now, there has been no literature available on the isolation of SARS-CoV-2 using initial in vivo and subsequent in vitro approach. Spike region of the virus isolated in hamsters revealed L212C amino acid change along with the C25000T nucleotide change compared to clinical specimens. A percent nucleotide difference of 0.0066% was observed, indicating a minimal change of Omicron sequence in Vero CCL-81 P-1 with hamster P-2. Nonetheless, a recent study conducted by VanBlargan et al. reported that even a single mutation such as R346K might be critical for neutralization escape. The possibility of uncontrolled mutations will be difficult to account . A detaiApparently, the sequences of human clinical specimens, hamster specimens, and Vero CCL-81 passages did not show any mutations in the furin cleavage site during sequential passages in different host species. Such virus isolate would retain its pathogenicity as per the original specimen/isolate. The isolated Omicron virus would be useful in new vaccine development, vaccine efficacy studies, pathogenicity studies, antiviral research, and estimation of virus neutralizing titers in vaccinated and recovered individuals.


To the editor.Herpesviruses, including herpes simplex virus 1 (HSV-1) and varicella-zoster virus (VZV) are commensal viruses of humans since millenaries.Close to 80% of adults in temperate countries are exposed to HSV-1 and this proportion reaches more than 97% in case of VZV [After primoinfection these viruses remain in a latent form in sensory neuronal ganglia. The colonisation of the ganglia, including the trigeminal ganglion, occurs by progression of the virus mainly within peptidergic neurons . LatencyWe recently examined a 53-year-old man, treated in the past for herpes kerato-uveitis OD that had been inactive for 18 months without treatment. Five days after the second dose of Moderna vaccine, he presented with a severe flare-up of a granulomatous hypertensive uveitis in his right eye with numerous granulomatous KPs and an increased pressure of 41 mmHg (Fig. It is difficult to know whether this episode was purely inflammatory. Even if this was the case dual corticosteroid and antiviral therapy has to be given in such circumstances.Since this case, we advise patients having had several herpes uveitis episodes to remain under valacyclovir therapy during vaccination despite quiet uveitis to avoid potential reactivation, which was undertaken for two patients so far.HSV-1 and VZV infections can cause substantial ocular morbidity. Since the availability of efficient antiviral agents, management of eye disease caused by herpes viruses are successfully treated in most cases and complications have been reduced . However