Additionally, costs incurred for ICU increased significantly in 2020 relative to those incurred in 2019 for both AGHA  and PUH . Hospitalisation costs for AGHA also increased significantly  in 2020 versus those obtained in 2019; however, no significant differences were found for PUH  in the same period analysed. However, the number of patients treated in the areas of HES, hospitalisation, and ICU was significantly reduced throughout 2020 compared to 2019. (4) Conclusions: Our findings show that the costs incurred during 2020 in the regional hospitals of Andalusia (Spain) increased significantly in most of the parameters analysed relative to those incurred in the year before the pandemic .(1) Background: The global health crisis caused by the coronavirus disease (COVID-19) pandemic has led to extreme overloading of different public healthcare systems worldwide. The Spanish Public Healthcare System is one of them. This study aimed to conduct a comparative cost analysis to assess the impact of the COVID-19 pandemic on small- and medium-sized regional hospitals in Andalusia (Spain). (2) Methods: This comparative, multicentre, observational, and retrospective study was designed to perform a comparative cost analysis between the Alto Guadalquivir Health Agency (AGHA) and Poniente University Hospital (PUH), both of which belong to the Spanish Public Health System (PHS). The data included in this study corresponds to the total costs by area and year incurred by the 61,335 patients from both healthcare institutions  in the areas of hospital emergency service (HES), hospitalisation, and intensive care unit (ICU), during the 24 months of the study period (from 1 January 2019 to 31 December 2020). (3) Results: The analysis results showed a significant increase in costs incurred in 2020 for HES relative to those incurred in 2019 for both AGHA (+14%;  Since the Chinese health authorities notified the World Health Organization (WHO) about the first case of an unknown type of pneumonia more than 2 years ago, the lives of millions of people worldwide have changed dramatically ,2,3,4. WSpain’s first SARS-CoV-2 case was reported on 30 January 2020 . Since tSince the emergence of COVID-19, a multitude of studies related to different aspects of this disease have been conducted . HoweverThis comparative, multicentre, observational, and retrospective study was designed to perform a comparative analysis of costs in small- and medium-sized regional hospitals located in central and eastern Andalusia (Spain).The 9 healthcare centres participating in this study were as follows: (1) Alto Guadalquivir Health Agency is responsible for healthcare management in the provinces of Jaen and Cordoba . It serves a reference population of 238,856 inhabitants, distributed in 8 healthcare centres, as follows: 62,128 correspond to the Alto Guadalquivir Hospital in Andújar, 61,687 to the Montilla Hospital, 24,114 to the Sierra de Segura High Resolution Hospital, 30,048 to the Puente Genil High Resolution Hospital, 10,498 to the Alcaudete High Resolution Hospital, 23,143 to the Valle del Guadiato High Resolution Hospital, 23,143 to the Alcalá la Real High Resolution Hospital, and 27,238 to the Sierra de Cazorla High Resolution Hospital . (2) TheBetween October 2020 and June 2021, total costs by area and year incurred by the 61,335 patients from both healthcare institutions  in the areas of hospital emergency service (HES), hospitalisation, and intensive care unit (ICU) during the 24 months of the study period (from 1 January 2019 to 31 December 2020) were included. The data on the generated economic costs were obtained from the Economic Management Units of both healthcare institutions.Direct costs from the hospital perspective were calculated in euros (year 2020). Both healthcare institutions belong to the Spanish Public Health System. Discounts and indirect costs were not considered in the calculation. Total costs by area and year in the areas of hospitalisation, intensive care unit, and hospital emergency services (HES) were included in this study. The costs of laboratory tests (biotechnology), maintenance/cleaning, human resources, material resources, and imaging studies (radiography) utilised during the stay of patients in the previously mentioned areas were also accounted such as ’type of cost and year’. Indirect costs were not included in the analysis because it requires imputation techniques, which could distort the comparison between different hospitals (in addition to the difficulty in obtaining this information).The study protocol was approved by the Regional Ethics Committee for Health Research (CEIC-AL: 91/2020). The present study was conducted under the precepts of the Declaration of Helsinki  and Spant-test was used for dependent samples with a normal distribution; otherwise, the Wilcoxon signed-rank test was used. For a comparison between the healthcare institutions, Student’s t-test was used for independent samples with a normal distribution; otherwise, the Mann–Whitney U test was used. p-value was estimated using the Wilcoxon signed-rank test for dependent samples comparing both institutions for the two years. Similarly, nonparametric tests were performed to compare the type of cost in both healthcare institutions for the two years. The Bonferroni correction method was used to control the ‘overall significance level’ of the set of comparisons performed. All statistical analyses were performed using the free software program R version 4.0.5 ; p ≤ 0.05 was considered to indicate statistical significance.First, a descriptive analysis was conducted. The cost variable was considered as a quantitative variable. Therefore, measures of central tendency  were calculated by month and for the entire year. The normality of the quantitative variable was tested with the Kolmogorov–Smirnov test. To test the hypothesis of equality of location measures for different intra-hospital years, Student’s p = 0.012) in 2020 relative to 2019 (p = 0.003), compared to 2019. Moreover, emergency service costs were 14% higher than those incurred in the year before the pandemic, that is, in 2019 (p = 0.003). As shown in The costs of hospitalisation in AGHA increased significantly ( to 2019 . As for p = 0.002) relative to those in 2019. Additionally, emergency service costs were 36% higher in 2020 than in 2019. As shown in The results of the analysis of the data collected from PUH  showed tp = 0.003), passing through 21% for X-ray (p = 0.006), 23% for material resources (p = 0.05), and finally 33% (p = 0.003) for biotechnology. On the other hand, despite having increased costs by EUR 1993, there were no significant differences with respect to the previous year. As shown in In AGHA, a significant increase was noted in costs associated with most of the parameters in 2020 relative to those obtained in 2019 . The ecop = 0.008), passing through 67% in biotechnology (p = 0.008), with 98% reached in material resources (p = 0.005) to, finally, a very significant growth of 127% (p = 0.005) reached in maintenance/cleaning. However, despite the 18% increase in costs for radio-diagnostic tests (radiography), no significant differences were noted when compared with the costs incurred in 2019 (p = 0.06).In the case of PUH, there was a very significant increase in costs associated with most of the variables in 2020 relative to those obtained in 2019 . The incp = 0.000). As for the type of cost, significant differences were noted in all the parameters except in the costs incurred for human resources (p = 0.821). As shown in As shown in To the best of our knowledge, this has been the first multicentre study to present the results of a comparative analysis performed with the main objective of quantifying the increase in costs, in general terms, associated with the emergence of the pandemic caused by COVID-19 in small- and medium-sized regional hospitals (AGHA and PUH) in Andalusia (Spain).The comparative analysis of costs showed that in 2020 (relative to 2019), there was a significant increase in the average costs in the three areas analysed , except in hospitalisation of PUH in that, although the average costs increased by EUR 91,098, they were not significant.As for the average annual number of patients attended, a significant reduction was noted in both hospitals. The reduction was significant in the emergency departments of both hospitals. The number of hospitalised patients was found to be reduced in both centres, with the reduction being significant only in PUH. The average number of patients admitted to the ICU was significantly reduced in AGHA in 2020. In contrast, in the data obtained for PUH, the average number of ICU patients increased in 2020 compared with that observed in 2019; however, this increase was not significant.The mean total cost per patient increased significantly in the three analised areas of all the centres participating in this study. The mean bill amount per hospitalised patient (AGHA = EUR 905.26 versus PUH = EUR 1605.38) increased significantly in both institutions (no distinction is madebetween patients with or without COVID-19); this value was lower than that reported in literature in previous studies on COVID-19 patients ,37,38. AAs for the comparative analysis conducted according to type of cost and year, a generalised increase was seen in the average annual costs in all the parameters evaluated. The increase in average costs was significant in the areas of biotechnology, human resources, and material resources in both hospitals. As for maintenance/cleaning, the increase in average costs per year was significant only in PUH. In contrast, the average annual costs associated with the performance diagnostic tests (radiography) showed a significant increase only in AGHA.The average costs by area were higher for hospitalisation (AGHA: +9% versus PUH: +6%) and ICU in PUH (+46%) than those for AGHA (30%). However, the average costs for the emergency departments were higher in AGHA than in PUH. The differences in the average costs per area between both medical hospitals were significant in the hospitalisation and emergency services areas.A comparison of the average costs by type of cost showed that the costs were higher for human resources, material resources, and diagnostic tests (radiography) in AGHA, with significant differences in the last two parameters, than those in PUH. In PUH, the average costs were higher for biotechnology and maintenance/cleaning, with significant differences in both hospitals when compared with those in AGHA.This comparative analysis of healthcare activity had some methodological weaknesses and strengths that need to be taken into account.First, the data provided by both PUH and AGHA were not disaggregated; hence, it was not possible to perform a comparative economic analysis among patients diagnosed with COVID-19. This limitation indicates that the results presented in this study could not be compared with those of previous studies because most of the available publications have shown results centred on COVID-19 patients.Second, the costs associated with areas other than hospitalisation, ICU, and emergency care, such as pharmacy, were not included in the analysis, which limits the generalisability of the study results.Third, this study was conducted from the perspective of the Andalusian Public Health System, belonging to the Spanish Public Health System. For this reason, it was not possible to perform an analysis of indirect costs from patients’ perspective, and it was not possible to estimate informal costs such as travel expenses and/or loss of work productivity of patients and/or accompanying persons, because it was not possible to interview the patients. Therefore, the economic impact would have been very different.The fourth limitation and coinciding with the finding of a previous study  is the rThe strengths of this study are directly related to the description of the comparative economic analysis performed. The study provides relevant information on the significant increase in the budget attributed to hospital care activity as a consequence of the emergence of COVID-19 in small- and/or medium-sized regional hospitals in Andalusia (Spain) when compared with that in the year before the pandemic. In addition, the Bonferroni correction method was used to control the ‘overall significance level’ of the set of comparisons performed.These results can be used by public health service managers to further investigate the clinical impact of COVID-19 in areas not included in this study. Additionally, future cost–opportunity studies could reveal the indirect costs borne by patients and public healthcare systems owing to the reduction in hospital care activity caused by COVID-19.The results of this study show that the costs incurred during 2020 in the regional hospitals of Andalusia (Spain) increased significantly in most of the parameters analysed relative to those in the year before the pandemic. The number of patients treated in HES, hospitalisation, and ICU was found to be significantly reduced during the first year of the pandemic. Additionally, the periods in which there was a greater increase in expenditure coincided with the peak periods of the first wave (March–June) and the second wave (in the last quarter of the year) of COVID-19 in Spain.


The objective of the study was to discuss the classification, virus characteristics, detection methods, and control measures of chickpea virus, with an aim to provide a theoretical basis for identification of new chickpea virus and control of chickpea virus disease.  The domestic and foreign studies were reviewed, and the virus coat protein or nucleic acid sequence was identified by immunological and molecular diagnostic techniques.  There were 14 main types of chickpea viruses attacking, and seven Luteoviridae viruses were reported, namely, chickpea chlorotic stunt virus (CpCSV), bean leafroll virus (BLRV), beet western yellows virus (BWYV), soybean dwarf virus (SbDV), cotton leafroll dwarf virus (CLRDV), cucurbit aphid-borne yellows virus (CABYV), and phasey bean mild yellows virus (PhBMYV). The family Geminiviridae includes chickpea chlorotic dwarf virus (CpCDV), chickpea chlorosis virus (CpCV), chickpea redleaf virus (CpRLV), chickpea yellows virus (CpYV), and mastrevirus. The family Nanoviridae is dominated by the faba bean necrotic yellows virus (FBNYV). The family Bromoviridae includes cucumber mosaic virus (CMV) and alfalfa mosaic virus (AMV).  At present, there are mainly 12 types of viruses infecting chickpeas, which are transmitted by leafhoppers or aphids and are associated with symptoms such as yellowing, chlorosis, and stunted pod development, resulting in serious yield loss. Correct use of various molecular diagnostic tools to detect and identify chickpea virus can accurately assess chickpea virus infection and provide a basis for the prevention and treatment of chickpea virus disease. Cicer arietinum L.) belongs to the genus Chickpea in the family Leguminosae, with a long history of cultivation, and first appeared in western Asia and the Near East. It is now mainly distributed in the Mediterranean, Asia, Africa, and America [Chickpea . It can damage the bast, and chickpea is severely stunted and slightly discolored after infestation, with young leaves curled, incompletely developed, thickened, and brittle leaves, and interveinal symptoms such as faded green spots, smaller young leaves that cup upwards, while mature leaves curl downwards, and stunted new shoots, leaves, and flowers [The family Nanoviridae is dominated by the genus faba bean necrotic yellows virus (FBNYV), which has a wide range of virus hosts and has been identified to infest more than 50 species of plants (mainly belonging to the legume family) , 42. Thi flowers , 43, 44. flowers .μm and lengths of 58 μm, 49 μm, 38 μm, and 29 μm, respectively; the other is a subspherical body with a diameter of about 18–20 μm. The nucleic acid accounts for about 18% of the weight of the viral particle, and the capsid protein occupies 82% [The family Bromoviridae, which includes cucumber mosaic virus (CMV) and alfalfa mosaic virus (AMV), is distributed in temperate and tropical areas with an extremely wide host range . The virpies 82% .CMV was first reported by Doolittle and Jagger in 1916 , while Chickpea virus disease seriously compromises the yield of chickpeas. Accurate and rapid identification and detection of the chickpea virus can effectively mitigate the harm. In the early stage, the virus was mainly identified by growth detection or direct observation, and the infecting virus was identified directly through observation of the symptoms of infected plants. The method is simple and cost-effective but susceptible to environmental factors. Given the similar symptoms of chickpea virus infestation, accurate identification of the viruses is complicated. Immunological and molecular diagnostic techniques can accurately identify viral coat proteins or nucleic acid sequences, thereby accurately identifying virus species. The techniques currently used in the detection of chickpea virus include serological or immunological detection, polymerase chain reaction, and high-throughput sequencing technology.Serology technology is an immunological technology, which uses antigen-antibody-specific binding in vitro for identification. This method is simple to operate with accurate results and is widely used in plant virus detection. According to the principle of color development, it is divided into enzyme-linked immunosorbent assay (ELISA) see  and immuMolecular diagnostic techniques allow the determination of viral nucleic acid sequences and thus the identification of viral species. The techniques are simple, sensitive, specific, and effective for the detection of low-load viruses. Polymerase chain reaction (PCR) is effective in detecting and identifying DNA viruses   Figure , and revThe aforementioned methods can detect plant viruses at a pg or even fg level, but differences are found in sensitivity between methods. Rowhani et al. used RT-PCR, IC-PCR, and ELISA to detect ApMV (apple mosaic virus), PDV (prune dwarf virus), PNRSV (prunus necrotic ringspot virus), GFLV (grapevine fanleaf virus), and CLRV (cherry leafroll virus) and found the highest sensitivity of RT-PCR, followed by IC-PCR, while ELISA was the least sensitive . HoweverThe control of plant viral diseases requires consideration of the “plant host-pathogen-environmental factors” and their interconnection to effectively control virus infections and losses see . CurrentEnhanced disease resistance in chickpea can reduce the damage caused by viruses, such as the selection of disease-resistant plants and the use of various factors to induce the production of disease resistance . CurrentThe first is to screen disease-resistant varieties. Comparative trials were conducted on different chickpea varieties to screen out different varieties with high fresh grass yield, high seed yield, strong adaptability, drought, and disease resistance to improve the disease resistance of chickpea. For example, the varieties FLIP94-68C, FLIP94-93C, and FLIP94-80C with excellent traits were screened in the Gansu area, which have strong disease and drought resistance and can be promoted in arid or semiarid areas .The second is crossbreeding, where genetic recombination generates new genotypes and screens for new superior traits. Different chickpea varieties differ greatly in their resistance to BLRV, and crossing between more distantly related varieties to screen for resistant monocultures in the progeny can significantly increase the proportion of resistant plants .The third is transgenic breeding, where antisense sequences of target virus gene fragments are transformed into plants to trigger the host plant to develop resistance to this virus through a gene silencing mechanism. For example, transgenic fava beans resistant to FBNYV infection effectively reduce the risk of plant infection with this virus and reduce disease losses .The control of the large-scale occurrence of virus diseases requires both intervention in the plants and reasonable ways to cut off the virus transmission channels and minimize the damage caused by virus diseases. Twelve chickpea viruses are transmitted by leafhoppers or aphids, so controlling the number of leafhoppers or aphids can effectively reduce the damage caused by chickpea virus diseases. Therefore, aphids and leafhoppers should be trapped and deterred regularly to lower the risk of plant infection; diseased plants should be cleaned in time to eliminate the pathogen, and operators, farm tools, and machinery should be disinfected to eliminate potential viruses , 65.Measures such as controlling the sowing period, sowing volume and row spacing, inducing disease resistance early in the growing season, and cultivating disease-resistant and early-maturing varieties are effective in reducing virus infection in the field. Pretreatment of seeds with 1-2 insecticide sprays or broad-spectrum insecticides before planting can be effective in reducing virus transmission and virus disease incidence . Virus dCurrently, there are 12 major viruses that infest chickpea and are transmitted by leafhoppers or aphids, causing symptoms such as yellowing and greening, stunting, and pod stunting, which cause severe losses. Proper detection and identification of chickpea viruses using various molecular diagnostic tools can accurately assess chickpea virus infection and provide a basis for chickpea virus disease control. To reduce the damage of chickpea virus disease, chickpea virus disease control measures should be developed according to local conditions, including chickpea disease resistance improvement, virus transmission pathways reduction, and integrated virus management, so as to reduce the economic losses caused by virus infection.In recent years, the area planted with chickpea in China as well as the risk of occurrence and spread of chickpea virus disease has been increasing, and scientific prevention and management are urgently needed to avoid serious yield loss. Xinjiang is the largest area of chickpea cultivation in China, effectively promoting the realization of precise poverty alleviation in the region. Prevention and control of chickpea virus disease is an important measure to guarantee a good chickpea harvest; therefore, specific research on chickpea virus disease species identification, detection means, transmission routes, and virus transmission should be strengthened to develop specific and effective chickpea virus control measures in Xinjiang and safeguard chickpea cultivation.


Discoviridae were the highest. Surprisingly, the RDaBV NSs protein did not match any viral proteins. Phylogenetic analysis based on RdRp indicated that RDaBV is evolutionarily close to viruses in the family Discoviridae. The PVX-expressed system indicated that RDaBV N and NS may be symptom determinants of RDaBV. Our movement complementation and callose staining experiment results confirmed that RDaBV NSs is a viral movement protein in plants, while an agro-infiltration experiment found that RDaBV NS is an RNA silencing suppressor. Thus, we determined that RDaBV is a novel rice-infecting bunya-like virus.Bunyaviruses cause diseases in vertebrates, arthropods, and plants. Here, we used high-throughput RNA-seq to identify a bunya-like virus in rice plants showing the dwarfing symptom, which was tentatively named rice dwarf-associated bunya-like virus (RDaBV). The RDaBV genome consists of L, M, and S segments. The L segment has 6562 nt, and encodes an RdRp with a conserved Bunya_RdRp super family domain. The M segment has 1667 nt and encodes a nonstructural protein (NS). The complementary strand of the 1120 nt S segment encodes a nucleocapsid protein (N), while its viral strand encodes a small nonstructural protein (NSs). The amino acid (aa) sequence identities of RdRp, NS, and N between RDaBV and viruses from the family  Bunyavirales [Arenaviridae, Cruliviridae, Fimoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, and Wupedeviridae, and two new families  created by ICTV in July 2021 [Penaeus chinensis) on a farm in China [Euproctis pseudoconspersa population obtained from the field [Bunyaviruses are often transmitted by vectors and can infect vertebrates, arthropods, and/or plants . In 2018uly 2021 . Virionsuly 2021 . Genomesuly 2021 . The L suly 2021 ,7. The Suly 2021 ,7. As thuly 2021 ,10. For in China . AnotherUnlike other microorganisms, viruses cannot be analyzed using any universal conserved sequences, which makes it difficult to identify a novel virus . In receOryza sativa) is one of the most important food crops in the world and is planted mainly in Asian countries, including China [Rice . The RDaBV genome consists of L, M, and S segments. A phylogenetic analysis using RdRp amino acid sequences indicated that RdaBV is evolutionarily close to viruses from the family http://rice.plantbiology.msu.edu/, accessed on 13 October 2019). The none-plant specific reads were then de novo assembled using the Trinity v2.3.2 software [During the surveys of rice viral diseases in 2019, rice plants showing dwarfing, reduced tillering, and blighted grain were found and sampled from rice paddies in Zhejiang province, China. Total RNA was isolated from these rice samples using TRIzol reagent . The extracted total RNA samples were used for cDNA library constructions, which were performed by the Zhejiang TianKe High-Technology Development Co., Ltd. , followed by sequencing on an Illumina HiseqTM 4000 sequencing system , as previously described . Low-quasoftware .For viral genome amplification, total RNA was extracted from the collected rice samples and reverse-transcribed into cDNAs using the reverse transcriptase M-MLV  according to the manufacturer’s instructions. Super-fidelity PCR was performed using the KOD One PCR Master Mix  and specific primers . The rapTo survey viral infections in the paddy fields, total RNA from field rice plants was reverse-transcribed using the ReverTra Ace qPCR RT Master Mix with gDNA Remover  according to the manufacturer’s instructions. PCR reactions were performed to detect RDaBV using the Green Taq Mix  and the specific forward and reverse primer pair, RDaBV-Detection-F/R . Each PChttp://www.ncbi.nlm.nih.gov/orffinder, accessed on 18 December 2019) and using the sequence of the Plasmopara viticola lesion-associated mycobunyavirales-like virus 8 as a model [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 10 January 2020) [Open reading frames (ORFs) in the viral genomic RNA of the new virus were predicted using the NCBI ORFfinder software ( a model . Three vry 2020) .Bunyavirales. Detailed information on these sequences is provided in https://www.lynnon.com/dnaman.html, accessed on 10 January 2021).For homology analyses of genomic RNAs or predicted proteins of the new virus, we downloaded the nucleotide and amino acid sequences of representative species from all fourteen families of the order Bunyavirales were downloaded from the GenBank database. Detailed information on these viruses is provided in https://itol.embl.de/, accessed on 10 January 2021).Genome sequences of representative viruses in different families of the order NS, N, and NSs protein genes of the newly identified virus were individually amplified from the diseased rice plants with RT-PCR using the specific primers listed in Agrobacterium tumefaciens strain EHA105. Leaves of line 16c transgenic Nicotiana benthamiana were co-infiltrated with A. tumefaciens harboring the GFP gene and A. tumefaciens harboring the N, NSs, NS, tomato bushy stunt virus (TBSV) P19 or GUS gene, respectively. GFP fluorescence in co-infiltrated leaves was observed and photographed under a UV lamp .To identify the RNA silencing suppressor of RDaBV, the constructed vectors were confirmed by nucleotide sequencing and then transformed into the N, NSs, and NS were separately subcloned into the PVX heterologous expression vector pGR106. The resulting recombinant expression vectors were transformed into the A. tumefaciens strain GV3101 using the freeze–thaw method as previously described [N. benthamiana leaves were infiltrated with A. tumefaciens harboring the PVX-base expression vector. At 7 days post-agro-infiltration (dpi), the agro-infiltrated N. benthamiana and its leaves were observed and photographed.To identify the symptom determinants, RDaBV escribed . N. bentA. tumefaciens strain GV3101 harboring the vector PVX-GFPΔp25 was prepared and diluted to OD600 = 0.001 in an infiltration buffer  and 100 μM acetosyringone), as previously described [v/v/v) with A. tumefaciens cells harboring a vector expressing the TBSV P19 protein (OD600 = 0.6) and A. tumefaciens cells harboring a vector expressing the rice stripe virus (RSV)-encoded NSvc4 protein (OD600 = 0.6). For other treatments, A. tumefaciens harboring the vector expressing RSV NSvc4 was replaced by A. tumefaciens harboring the vector expressing the new virus NS, N, or NSs protein, or A. tumefaciens harboring the vector expressing β-glucuronidase (GUS). The mixed A. tumefaciens cells were infiltrated into N. benthamiana leaves. At 6 dpi, the infiltrated leaves were examined under a UV light or a confocal microscope.An expression vector expressing a movement-defective potato virus X  was obtained from a previously published source . The A. escribed , and theN. benthamiana leaves was extracted in a protein extraction buffer [The total protein of n buffer , followen buffer . In brieField-collected rice plants were analyzed for viral infections using RT-PCR, after which leaf tissues were sampled from the diseased rice plants and fixed for 4 h in a 2.5% glutaraldehyde solution (pH 7.0) at room temperature. The fixed leaf samples were dehydrated in serially diluted ethanol solutions. After 20 min of incubation in acetone, the tissues were individually embedded in the Spurr resin . Ultra-thin sections were cut from the embedded samples and examined under a Hitachi H-7650 transmission electron microscope.N. benthamiana plants and observed under a confocal microscope . The excitation wavelengths of GFP, RFP, and aniline blue were 488, 555, and 405 nm, respectively, and the emission wavelength was set at 510 nm for GFP, 583 nm for RFP, and 461 nm for aniline blue.The agro-infiltrated leaves were cut from the Bunyavirales. These three long contigs corresponded to the large (L), medium (M), and small (S) segments of bunyaviruses, respectively. To confirm this RNA-seq result, we amplified partial segments of the above three long contigs using RT-PCR, and all gene segments could be amplified  showed that these leaf samples contained enveloped spherical virus-like particles approximately 125 nm in diameter E, similadiameter  and the diameter .Discoviridae  and a nucleocapsid (N) protein [Discoviridae ranged from 30.53 to 50.17% and was much higher than that between RDaBV and viruses of other families in the order Bunyavirales  in the order Mononegavirales in the NCBI database , we found that PVX-N and PVX-NS caused plant leaf necrosis symptoms, while PVX-NSs showed the same mosaic symptoms as PVX , pCambia 1300-N-GFP (N:GFP), and pCambia 1300-NSs-GFP (NSs:GFP). N. benthamiana leaves were individually agro-infiltrated with these expression vectors, and the green fluorescence was observed at 48 h post-agro-infiltration (hpi). The green fluorescence  was located at the cytomembranes, cytoplasm, and nuclei of N. benthamiana leaf cells, which is similar to that of GFP on its own , or the GUS expression vector (the negative control) were mixed with A. tumefaciens cells carrying the PVX-GFPΔp25 expression vector and A. tumefaciens cells carrying the TBSV P19 expression vector. The mixed A. tumefaciens cells were individually infiltrated into N. benthamiana leaves. At 5–7 dpi, the infiltrated leaves were harvested and examined under a confocal microscope and a UV lamp. The result showed that in the presence of NSs-flag, the mutant PVX-GFPΔp25 moved into the neighboring cells, similar to that observed in leaves co-expressing PVX-GFPΔp25 and RSV NSvc4 , FLAG-tagged N (N-flag), and FLAG-tagged NS (NS-flag). SV NSvc4 A,B. Thisa leaves A. We colSV NSvc4 C. The WeSV NSvc4 C. HoweveSV NSvc4 C.NSs-flag or GUS in N. benthamiana leaves. Two days later, the infiltrated leaves were harvested, stained with aniline blue, and examined under a confocal microscope. We found that leaves expressing NSs had fewer aniline blue-stained dots than those expressing GUS  shows the position of PDs in cell walls, our results provide support for the hypothesis that RDaBV NSs can target PDs  to inhibit virus cell-to-cell spread, while viral MPs can reduce callose depositions near PDs ,38. To fsing GUS A. We cousing GUS B. To invA. tumefaciens harboring a vector expressing GFP was mixed with A. tumefaciens harboring a vector expressing RDaBV NS-flag, N-flag, NSs-flag, P19, or GUS. The mixed A. tumefaciens combinations were then individually infiltrated into transgenic 16c N. benthamiana leaves. In this experiment, A. tumefaciens harboring the vector expressing GUS and P19 was used as the negative and positive control. At 6 dpi, the leaves co-expressing GFP and GUS, GFP and N-flag, GFP and NSs-flag, GFP and NS-flag, or GFP and P19 were harvested and photographed under a UV light. We found that GFP silencing was suppressed in the presence of RDaBV NS or P19 .It is well known that the number of known plant-infecting viruses accounts for a small fraction of the total number of plant-infecting viruses in nature. In our field survey in 2019, rice plants showing dwarfing, reduced tillering, and blighted grain symptoms, which are similar to that shown by virus-infected rice plants, were found in several rice fields in Zhejiang province, China. Because analyses of the collected rice samples for known virus infections through RT-PCR failed, we speculated that these rice samples could be infected with unidentified virus(es). To test this speculation, we analyzed these rice samples using RNA-seq. The RNA-seq and RT-PCR results showed that these dwarf rice plants contained an ambisense-segmented viral RNA genome, similar to that reported for viruses in the order Discoviridae in the order Bunyavirales. The RDaBV genome consists of three RNA segments: L, M, and S. The genome organization of RDaBV was determined according to the existence of conserved motifs and previously reported features of bunyaviruses [The organization of the RDaBV genome shares similar characteristics with that of viruses in the family aviruses ,2,3,4. Taviruses ,36.Laodelphax striatellus), brown planthopper (Nilaparvata lugens), white-backed planthopper (Sogatella furcifera), and Recilia dorsalis, but all failed (data not shown). A phylogenetic analysis using RDaBV RdRp showed that this novel rice virus belonged to the newly created family Discoviridae and was evolutionarily close to Plasmopara viticola lesions-associated mycobunyavirales-like virus 8 [Discoviridae are from the phylum Oomycota or Ascomycota. Given that we did not find the insect vector of RDaBV, we suspect that the novel virus could be transmitted by fungi but could also be transmitted by other insects.Though some plant viruses are diffused by contact and seeds , the maj virus 8 . The hosThe encoding of MPs is one of the most important characteristics of plant viruses. The plasmodesmata (PD) is the critical macromolecule transport corridor in plant cell walls. Because of its size exclusion limit (SEL), PD is a bottleneck during plant virus infections . Many stWe also found that the NS protein can act as a viral RNA silencing suppressor. RNA silencing (RNAi) is a common defense strategy used by plants to resist viral invasions. This anti-viral strategy can be interfered with by viral suppressors of RNA silencing (VSRs) encoded by plant viruses via inhibiting dsRNA recognition, interfering with RNA-induced silencing complex (RISC) assembly, directly binding to proteins crucial to the RNA silencing pathway, and interfering with silent signal transmission ,44,45,46Discoviridae of the order Bunyavirales in Zhejiang province, China. We determined that the RDaBV NSs protein is a viral movement protein and its NS protein is a viral RNA silencing suppressor. Since vectors play important roles in rice virus spread in fields, further studies are needed to determine how RDaBV is transmitted in rice fields. Furthermore, the pathogenicity of RDaBV must be verified using Koch’s postulate. Additionally, its evolutionary history and impact on rice production must be further studied. Nonetheless, the findings presented in this study enrich our understanding of bunyaviruses and rice viruses and can contribute to more effective strategies for controlling this rice viral disease.In summary, we identified a novel rice-infecting bunya-like virus, RDaBV, belonging to the family


Biothermodynamics of viruses is among the youngest but most rapidly developing scientific disciplines. During the COVID-19 pandemic, it closely followed the results published by molecular biologists. Empirical formulas were published for 50 viruses and thermodynamic properties for multiple viruses and virus variants, including all variants of concern of SARS-CoV-2, SARS-CoV, MERS-CoV, Ebola virus, Vaccinia and Monkeypox virus. A review of the development of biothermodynamics of viruses during the last several decades and intense development during the last 3 years is described in this paper. There is a common opinion that thermodynamics is a scientific discipline related to machines, engines and devices, dealing mostly with efficiency of energy transformation and utilization. Indeed, Lazarus Carnot ,2 and hiOften, the same researchers worked in the field of classical thermodynamics and biothermodynamics. Indeed, Boltzmann , one of Growth is one of the main characteristics of organisms. The answer to the question of what represents the driving force for the growth of organisms was given by von Stockar ,18,19,20Thermodynamic characterization of life processes has been a subject of interest for many researchers. Von Stockar et al. ,43 appliThe importance of thermodynamic considerations in life sciences is clearly seen from the Gibbs energy being used to define catabolic and anabolic processes . AnnamalViruses are the most abundant organisms: there could be more viruses than stars in the universe . There aWith the appearance of the COVID-19 pandemic, various scientific disciplines attempted to contribute, in the shortest time possible, to the fight against the pandemic. Molecular biology has played an important role with the reading of genetic sequences of SARS-CoV-2. Thermodynamics has joined the fight and in 2020, thermodynamic properties have been published for multiple viruses . An analInfectivity and pathogenicity are terms mostly used in microbiology, biology and medicine. These terms have their physical basis and driving forces in biothermodynamics. The basis of the infectivity of viruses is susceptibility and permissiveness . Antigen–receptor binding represents a chemical reaction, similar to protein–ligand interactions . The driTo explore the interaction between a virus and its human host, it was necessary to find thermodynamic properties for host organisms. Thermodynamic properties have been reported for human tissues ,105 sincThe second virus–host interaction is in the cytoplasm. In papers ,80, a biViruses represent the smallest organisms, but also belong to the most contagious and deadly microorganisms. They spread very rapidly, often causing epidemics and pandemics, which result in large numbers of casualties. Furthermore, there are very few antiviral medicines. Thus, the fight against epidemics and pandemics is directed towards epidemiological measures and the application of vaccines. However, vaccine production, especially in the case of new viruses, requires a lot of time and resources. For example, the vaccines against SARS-CoV-2 were awaited for a year. The ability of some viruses to develop mutations fast leads to the need for new vaccines. Some of the available novel vaccines have proved themselves effective for the Hu-1, Alpha, Beta, Gamma and Delta variants. However, these vaccines are much less effective for the newer Omicron variants due to their ability to evade the immune response. This has imposed a need for the production of polyvalent vaccines, which also takes time and long-term testing. Knowing the thermodynamic properties of the host and virus, as well as the application of a mechanistic model of interactions on the cell membrane and in the cytoplasm, could, in the future, contribute to designing new vaccines and antiviral medicines. Moreover, such knowledge could aid in finding places and methods for vaccine application. For example, every human tissue is characterized by a specific value of Gibbs energy of biosynthesis of its building blocks. On the other hand, every virus variant is characterized with its own specific Gibbs energy of biosynthesis. The ratio of these two values is the permissiveness coefficient, which is different for various virus–host cell pairs. The result of this is that some viruses can be synthetized in one type of cell, while in others their multiplication is significantly slower. By choosing a tissue for vaccine application where virus growth is slower, it is possible to give enough time to the immune system to respond to a low virus concentration. Such a vaccine would be attenuated (live), capable of inducing an immune response but, due to the low permissiveness coefficient, unable to cause a disease in a more severe clinical form. The attenuation process of a vaccine based on biothermodynamic properties would not be performed through a long passage that requires great resources and time, but through choosing a place of application where the virus can multiply very slowly. Thus, one of the potential applications of biothermodynamics in virology would be in vaccinology. Such a vaccine would not be based on empirical data but on engineering, using biothermodynamic tools, which would help to significantly save time and resources in the design and production of vaccines.The aim of this review paper is to summarize the intense development of viruses in the field of biothermodynamics during the last few decades and try to predict the directions of the future development of the youngest scientific discipline—virothermodynamics.This section discusses the methodologies used in biothermodynamics of viruses and the results they provide. First, the experimental techniques are discussed, followed by theoretical approaches.onk, dissociation rate constant, offk, and dissociation equilibrium constant, DK [DK [BK, standard Gibbs energies of binding, ΔBG⁰, binding phenomenological coefficients, BL, and binding rates, Br [The binding affinities of virus antigens to host cell receptors (susceptibility) have been measured using surface plasmon resonance ,119 and tant, KD ,119. SPRtant, KD . The nontant, KD ,121. It , KD [KD ,121. Theates, rB ,91.Calorimetry has been used to study viruses, including differential scanning calorimetry (DSC), isothermal titration calorimetry (ITC) and reaction calorimetry . Differential scanning calorimetry (DSC) measures the difference in heat absorption rates between sample and reference during gradual heating, revealing various thermal effects, such as phase transitions or protein unfolding ,123. DSCWhile DSC performs measurements by changing temperature, isothermal titration calorimetry (ITC) measures heat released or absorbed when a reagent is titrated into a solution at constant temperature ,123. ITCReaction calorimetry, or isothermal microcalorimetry, measures heat released or absorbed during a chemical reaction, usually at constant temperature (without titration like in ITC) ,123. Rea(1)Empirical formula;(2)Thermodynamic properties of live matter;(3)Thermodynamic properties of biosynthesis.The thermodynamic properties of viruses can be calculated using biothermodynamic methodology. Thermodynamic properties of virus biosynthesis can be found from virus elemental composition in three steps:The first step is to find empirical formulas of virus live matter. This can be achieved using the atom-counting method , which gr is the rate of a chemical process, T is temperature, while ΔG is Gibbs energy change of the process. L is a constant known as phenomenological coefficient, and is specific for each process. Phenomenological equations can be applied to both antigen–receptor binding and virus multiplication inside host cells [Br, and Gibbs energy of binding, ΔBG:BL is the binding phenomenological coefficient [Phenomenological equations are an important tool, relating thermodynamic and kinetic properties of processes ,178,179.st cells ,91. In tfficient ,91,180.bsr, to the Gibbs energy of biosynthesis, ΔbsG:bsL is the biosynthesis phenomenological coefficient [Similarly, the biosynthesis phenomenological equation relates the rate of biosynthesis of virus components, fficient ,95,180. fficient ,178.Experimental work with viruses can sometimes require high biosafety levels. However, there are few laboratories that work on calorimetric measurements that possess the appropriate biosafety level . Time, eThe path from thermodynamics to biothermodynamics was very short. The researchers who laid the foundations of classical thermodynamics were also the first to apply them to living organisms ,6. The r1.6390O0.2851N0.2301P0.0065S0.0038 [1.569O0.3281N0.2786P0.00173S0.00258 [In the introduction, it was mentioned that viruses represent the most abundant living organisms. Moreover, there is nearly 10 000 different virus species. However, during the last few years, empirical formulas have been determined for less than 50 species, while thermodynamic properties are known for less than 70 species. Various virus species (and variants) are characterized by specific empirical formulas. For example, the Hu-1 variant (wild type) of SARS-CoV-2 is characterized by its specific empirical formula CH5S0.0038 ,112. TheS0.00258 . This diS0.00258  or the aS0.00258 ,89,112.Panta rhei; the world is moving and changing. The natural driving forces are hidden in the objective world and the human body. What are the physicochemical forces that drive life? Organisms perform biological and chemical processes. The driving force of all chemical processes in animate and inanimate matter is Gibbs energy ,189,190.Viruses represent obligate intracellular parasites . Thus, tP, is given by the equationbsr represents the biosynthesis rate, while ΔbsG⁰ is standard Gibbs energy of biosynthesis [The permissiveness coefficient represents the ratio of rates of biosynthesis of virus components and host cell components. A permissiveness coefficient greater than one indicates the advantage in synthesis of virus components, leading to a successful viral life cycle inside the host. The permissiveness coefficient, ynthesis . A similynthesis . Basicalynthesis . This reynthesis .It is obvious that the biothermodynamics of viruses are able to offer a wide variety of important information, useful first of all to virologists, microbiologists, biologists, epidemiologists and infectologists. Knowing thermodynamic properties and mechanistic models that are developed in biothermodynamics can shed more light on basic processes from the domains of biophysics and chemistry, which represent the basis for biological phenomena. The immune response  implies antigen–antibody reaction. The antigen–antibody reaction is similar to protein–ligand interactions. Thus, the driving force for the antigen–antibody interaction is the Gibbs energy of this interaction. Cellular immune response is related to mobilization of immune cells and, thus, increase in number. This results in growth. Growth, like with other cells, represents a biological and biothermodynamic phenomenon, driven by Gibbs energy of growth. Thus, it is necessary to know the thermodynamic properties of immune cells. After an extensive search of the literature, the author could not find data on the thermodynamic properties of lymphocytes, leukocytes and macrophages. Infection is a complex biological process, which, except for the infective agent (microorganism), involves a host cell/tissue and immune cells. To reveal the thermodynamic basis of infections in full, it is necessary to know all 3 elements . Biothermodynamics is a young discipline and obviously faces many challenges and a great body of work that needs to be realized. The effort on the development of biothermodynamics seems justified since it can greatly help in explanation of pathogeneses of many diseases that occur as a result of microorganism–host interactions.Time evolution of viruses can be followed through change in Gibbs energies of binding and biosynthesis . VirusesBiothermodynamics of viruses is among the youngest scientific disciplines. However, appearance of new viruses, their rapid mutation, which can lead to epidemics and pandemics with a great number of cases and casualties, have given an impulse for the very rapid development of biothermodynamics of viruses. Knowing biothermodynamic properties can give useful information to epidemiologists and infectologists about the mechanism of virus–host interaction and virus–virus competition. Knowing empirical formulas of viruses is significant because it allows fast and accurate identification of known viruses or detection of new viruses or variants. Moreover, phenomenological equations, which belong to nonequilibrium thermodynamics, have proven themselves an important tool for analysis of rates of antigen–receptor binding and rates of virus multiplication inside host cells. The permissiveness coefficient could be useful during the estimation of the degree of damage to host tissues, caused by the multiplication of viruses, as well as the assessment of the outcome of virus–virus competition during the simultaneous presence of two viruses or virus variants in the same time and the same place.


Hard and soft surfaces (fomites) can play a role in the transmission of both enteric and respiratory viruses. Transmission can occur by touching of contaminated surfaces and bringing the hand to the either the mouth or nose or by re-aerosolization of the virus from the surface. In adults face touching occurs every 3 to 4 minutes. The amount of virus transfer which occurs depends on the nature of the surface, the virus and the degree and type of activity within a facility.We conducted studies on the finger transfers of both a non-enveloped (MS-2 coliphages) and an enveloped virus (coronavirus 229E).The greatest degree of the non-enveloped virus occurred with acrylic plastic and stainless steel, while the greatest transfer of the enveloped virus occurred from porcelain. Transfer to the finger from surfaces of the coronavirus was reduced by treatment of surfaces with various commercially available quaternary ammonium disinfectants after 24 hours of application to the fomites by 37% to 99.9%. Using MS-2 coliphage as a tracer virus placed on a high touch fomite, we found that the use of quaternary ammonium containing disinfecting wipes in a long term care facility reduced the contamination of fomites by 80%.The results indicate that spread of viruses in a facility and exposure via hand contact can be significantly reduced by disinfection of fomites.Charles P. Gerba, PhD, Allied Biosciences: Grant/Research Support|Boeing: Grant/Research Support|Corning: Grant/Research Support|GOJO: Grant/Research Support|Rickett: Advisor/Consultant|Rickett: Grant/Research Support.


Vitis labrusca × Vitis riparia), was successfully used to eliminate eight viruses (grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine Pinot gris virus (GPGV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine fleck virus (GFkV), grapevine rupestris vein feathering virus (GRVFV), grapevine Syrah virus-1 (GSyV-1), and raspberry bushy dwarf virus (RBDV)), as well as two viroids (hop stunt viroid (HSVd) and grapevine yellow speckle viroid 1 (GYSVd-1)) from preclonal candidates of six grapevine varieties (Vitis vinifera L.). A half-strength MS medium including vitamins supplemented with 30 g/L of sucrose and solidified with 8 g/L of agar, without plant growth regulators, was used for the growth and root development of micrografts and the subsequently micropropagated plants; no callus formation, hyperhydricity, or necrosis of shoot tips was observed. Although the overall regeneration was low (higher in white than in red varieties), a 100% elimination was achieved for all eight viruses, whereas the elimination level for viroids was lower, reaching only 39.2% of HSVd-free and 42.6% GYSVd-1-free vines. To the best of our knowledge, this is the first report of GPGV, GRVFV, GSyV-1, HSVd, and GYSVd-1 elimination through combining in vivo thermotherapy and in vitro meristem tip micrografting, and the first report of RBDV elimination from grapevines. The virus-free vines were successfully acclimatized in rockwool plugs and then transferred to soil.Viruses and virus-like organisms are a major problem in viticulture worldwide. They cannot be controlled by standard plant protection measures, and once infected, plants remain infected throughout their life; therefore, the propagation of healthy vegetative material is crucial. In vivo thermotherapy at 36–38 °C for at least six weeks, followed by meristem tip micrografting (0.1–0.2 mm) onto in vitro-growing seedling rootstocks of Vialla ( Viruses and virus-like organisms can cause severe economic problems in vitiviniculture due to (i) yield reduction, (ii) alterations of grape and wine chemical and sensorial quality, (iii) various developmental and morphological malformations in vine organs, and (iv) shortened vine life ,2,3,4. TSlovenia is a traditional wine-growing country, with 15,075 hectares of vineyards in 2021 . The Primorska wine-growing region represents 40.6% of the total Slovenian vineyard area, where a successful program of clonal selection, especially of indigenous, domesticated, and local grapevine varieties, has been taking place for decades. According to the Official Gazette of the RS N°93/05 and 101/20, all propagated vine material must undergo mandatory testing on: arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), raspberry ringspot virus (RpRSV), tomato black ring virus (TBRV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine leafroll-associated virus 1 and 3 , and grapevine fleck virus (GFkV) (only for rootstocks). Testing on grapevine leafroll-associated virus 2 and 4–9  is not obligatory but just recommended. In our previous study [Thus, in the present study, we report on the efficiency of in vivo thermotherapy followed by in vitro meristem tip micrografting in the elimination of the eight above-listed viruses (except GRGV) and two viroids from preclonal candidates of six grapevine varieties.A total of 598 meristems were isolated and micrografted, from which 51 plants were regenerated a,b. To iHigher regeneration rates were observed in white varieties compared with red varieties . Only onThe efficiency of the elimination of viruses and viroids from regenerated plants grown in vitro for seven months was analyzed using RT-PCR. A 100% elimination rate was achieved for all viruses. However, the elimination of viroids HSVd and GYSVd-1 was significantly lower at 39.2% and 42.6%, respectively . Specifi(Vitis vinifera L.), infected with various viruses and viroids, were included into the elimination process by in vivo thermotherapy followed by in vitro meristem tip micrografting.In this study, 28 preclonal candidates from 6 grapevine varieties According to Křižan et al. , in vivoThe elimination of phloem-limited viruses through the meristem tip culture is particularly effective, whereas thermotherapy, which hampers virus replication and promotes virus RNA degradation ,27, is dV. rupestris [GLRaV-3, the main causal agent of one of the most severe grapevine diseases, grapevine leafroll disease (GLD), is phloem-limited , somaticupestris ,47, wherupestris . Althougupestris , reportiupestris . It is nupestris ,6. A merupestris ,21. Salaupestris . In our upestris . In contupestris  reportedupestris . In contupestris  reportedupestris . It is aupestris . Gualandupestris  reportedupestris  reportedupestris , GPGV waupestris  and repeupestris . Previouupestris ,42,54 inupestris  reportedupestris ,16,19,42upestris  reportedupestris ,13,24,55upestris . Outsideupestris ,58,59,60upestris ,61,62,63Although the complete elimination of all eight viruses was achieved, sanitation rates for the widely distributed viroids HSVd and GYSVd-1 were much lower in our study, i.e., 39.2% and 42.6%, respectively, compared with virus elimination. Viroids accumulate at higher titers upon experiencing a high temperature; therefore, thermotherapy alone was unsuccessful in their elimination . DiffereIt can be concluded that the elimination success of viruses and viroids from vines depends on several factors. The generally high elimination rates and low regeneration rates found in our study could be because smaller meristems have low survival rates but higher efficiency in virus elimination. The low regeneration rate could be also linked to the fact that meristem isolation and micrografting techniques are difficult to handle and require a high level of expertise and rapid handling to avoid explant drying and oxidation problems. Overall, it is sufficient to obtain one virus-free, regenerated plant per candidate that can be further micropropagated.With somatic embryogenesis, high risks of somaclonal variation exist, whereas chemotherapeutics may prove highly phytotoxic. In our study, limiting the duration of the in vitro cultivation phase through in vivo thermotherapy and plant regeneration from meristems reduced the risk of genetic instability. However, regenerated plants will be carefully monitored. In addition, the elimination success rate using combined thermotherapy and meristem tip micrografting is encouraging. It remains to be seen whether our virus-free preclonal candidates will remain negative for up to about three years, when they will be retested prior to their official certification.Vitis vinifera L.): two reds, ‘Refošk’ (‘Terrano’) and ‘Pokalca’ (‘Schioppettino’), and four whites, ‘Laški rizling’ (‘Welschriesling’), ‘Rebula’ , ‘Malvazija’ , and ‘Zeleni Sauvignon’ (‘Sauvignon vert’). They were collected in February 2019, in the three vineyards  of the clonal center of Vrhpolje  where the clonal selection was conducted. However, preclonal candidates were selected in various vineyards in the Primorska region according to the rules on the marketing of material for the vegetative propagation of vines  and the OIV process for the clonal selection of vines (Resolution oiv-viti-564a-2017). Later, after vegetative propagation, they were planted in the vineyards of STS (45°50′02.2″ N 13°56′18.8″ E). One-bud cuttings were forced to bud burst and root in common tap water with no additional nutrients at room temperature (21–22 °C) at the Biotechnical Faculty, University of Ljubljana. After one month of rooting in water, well-rooted cuttings were planted into pots and transferred to the greenhouse, from which 28 plants were randomly selected for the sanitation study.Eighty-two woody cuttings of preclonal candidates, without exhibiting any visual morphological symptoms regarding viruses and viroids infections, were collected of the following grapevine varieties  were provided by INRAE, Montpellier, France. Seeds were surface-disinfected in a laminar flow hood with a 1.66% solution of sodium dichloroisocyanurate , supplemented with three drops of surfactant, Tween 20 , for 15 min with constant agitation. They were then washed three times with sterile, distilled water and plated (five seeds per plate)  a on a 1/r plate) b were ser plate) c.2/s in the growth chamber . The plant material obtained was micropropagated several times on a fresh medium with the same components.Thermotherapy was performed in a growth chamber  for a minimum of six weeks to a maximum of three months at a temperature of 36–38 °C, and a photoperiod of 16 h of light and 8 h of dark a. After 2 , 1.6 μL of dNTP mix (10 mM of each of the 4 dNTPs) (Promega), 0.5 μL of each primer, 0.1 μL of KAPA Taq DNA polymerase (Kapa Biosystems), and 1 μL of cDNA. The primers used for testing after the sanitation experiment are listed in Virus and viroid elimination rates were determined by examining the tissues of plants maintained in vitro for seven months. Total RNA was extracted from all regenerated plants using the Monarch RNA Total Miniprep Kit (New England Biolabs). RNA concentration, quality, and purity were checked using the Agilent 2100 Bioanalyzer  and NanoVue Plus Spectrophotometer . cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), following the manufacturer’s instructions. The PCR was performed in a 20 μL reaction volume containing 10.7 μL of nuclease-free water, 4 μL of 5× PCR buffer , 1.6 μL of MgClA few virus-free and well-developed in vitro plants per preclonal candidate were taken out of the tissue culture jars and washed with sterilized water to remove any adherent medium. They were then transferred to mini greenhouses in rockwool plugs with added perlite (Plagron) and kept in a growth chamber  for two months at 25 °C with a photoperiod of 16 h of light and 8 h of darkness. During acclimatization, the plants were irrigated with MS including vitamins (Duchefa Biochemie). The vents on the mini greenhouse covers were gradually opened. The acclimatized plants were later transplanted into pots and cultivated under greenhouse conditions at the STS.


Paraglaciecola Antarctic GD virus 1 (PANV1), Paraglaciecola Antarctic JLT virus 2 (PANV2), Octadecabacter Antarctic BD virus 1 (OANV1), and Octadecabacter Antarctic DB virus 2 (OANV2). The phages infect common sea ice bacteria belonging to the genera Paraglaciecola or Octadecabacter. Although the phages are marine and cold-active, replicating at 0°C to 5°C, they all survived temporal incubations at ≥30°C and remained infectious without any salts or supplemented only with magnesium, suggesting a robust virion assembly maintaining integrity under a wide range of conditions. Host recognition in the cold proved to be effective, and the release of progeny viruses occurred as a result of cell lysis. The analysis of viral genome sequences showed that nearly one-half of the gene products of each virus are unique, highlighting that sea ice harbors unexplored virus diversity. Based on predicted genes typical for tailed double-stranded DNA phages, we suggest placing the four studied viruses in the class Caudoviricetes. Searching against viral sequences from metagenomic assemblies, we revealed that related viruses are not restricted to Antarctica but are also found in distant marine environments.Although we know the generally appreciated significant roles of microbes in sea ice and polar waters, detailed studies of virus-host systems from such environments have been so far limited by only a few available isolates. Here, we investigated infectivity under various conditions, infection cycles, and genetic diversity of the following Antarctic sea ice bacteriophages:  Sea ice covers a significant area of polar oceans every year, affecting ocean ecology, biogeochemical cycles, and climate , 2. Sea 3–9–12–Shewanella, Flavobacterium, Colwellia, Octadecabacter, Glaciecola, and Pseudoalteromonas strains , at 50°C, 55°C, 40°C, and 50°C for PANV1, PANV2, OANV1, and OANV2, respectively  . The stuectively .2+, even if residual Mg2+ ions were removed by the chelating agent ethylenediaminetetraacetic acid (EDTA)  buffer, the OANV1 titer dropped by 4 to 5 orders of magnitude, and the OANV2 titer dropped to 1/10th (2+ ions were removed using EDTA (4 PFU/mL) and the OANV2 titer dropped 100-fold. When both NaCl and MgSO4 were removed (4 PFU/mL.All four viruses studied here remained infectious without NaCl (2PO4 (see 4 PFU/mL).No statistically significant difference in virus titers was observed when virus stocks were incubated in SM buffer (pH 7.5) with either Tris-HCl or NaH2PO4 see  to h. PA10.1128/mbio.00651-22.6FIG S12PO4 (buffer 1) or Tris-HCl . Virus stocks were diluted 1,000-fold in the buffers and incubated 1 h (dark gray) and 5 h (light gray). Bars represent means of at least three independent replicates with standard error of the mean shown as error bars. Download FIG S1, PDF file, 0.1 MB.Stability of virus infectivity in SM buffer containing NaHCopyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the k calculated for the first 30 min p.i. (n = 3) was 3.9 × 10−9 and 9.0 × 10−12 mL/min for PANV2 and OANV2, respectively. PANV1 and OANV1 adsorbed with the rates of 5.4 × 10−10 and 4.6 × 10−13 mL/min, respectively, reaching ~70% adsorption by 12 h p.i.PANV1, PANV2, OANV1, and OANV2 adsorbed effectively to their hosts, achieving at least ~50% binding efficiency within 6 h at 4°C . During Paraglaciecola IceBac 372 and Octadecabacter IceBac 419 and 430 grew to early stationary stage in 4 to 7 days , showing typical growth curves of a bacterial culture. The cultures were infected at the logarithmic growth phase  using a multiplicity of infection (MOI) of 8 to 10 to analyze the one-step growth of the phages  reduced almost 2 orders of magnitude as a result of the virus addition (~2.3 × 107 CFU/mL) by 124 h p.i., while the number of viable cells in the uninfected culture of IceBac 419 was growing (~3.3 × 109 CFU/mL by 124 h). This result can be interpreted as lysis being caused by the virus infection. Similarly, the numbers of viable cells in the OANV2-infected IceBac 430 culture dropped noticeably (from ~1.4 × 109 at 0 h p.i. to ~5 × 106 CFU/mL at 124 h p.i.) compared with those of the uninfected culture (~3.5 × 109 CFU/mL at 124 h p.i.).Bacterial strains e phages . Uninfec4 h p.i. . The tur4 h p.i. . For PANPANV1, PANV2, OANV1, and OANV2 genomes are double-stranded DNA (dsDNA) molecules ranging from ~36 to ~151 kb, with GC content of 38% to 62% and 61 to 243 predicted protein-coding open reading frames (ORFs) . All fou10.1128/mbio.00651-22.1TABLE S1Table S1, PDF file, 0.3 MB.Putative functions assigned to PANV1 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The majority of predicted ORF products (63% to 71%) of the four virus genomes could not be assigned with any functions, including unique sequences (43% to 48%) that had no homologues in the NCBI nonredundant (nr) protein database . SurprisAbout 29% to 37% of the studied sequences had homologs in the database, allowing us to predict their functions. BLAST matches revealed mosaic similarities to the sequences of other phages, as well as moderate and psychrophilic bacteria. The following functional categories were assigned to the gene products (gps): (i) proteins involved in DNA replication, recombination, modification, and metabolism; (ii) transcription and translation regulation proteins; (iii) virion and tail structural components; (iv) cell lysis; and (v) other functions ; Fig. 5.Bordetella phage BPP-1 bbp10, which is a lysin containing a beta/gamma crystalline motif . In PANV1, gp232 and gp233 were annotated as baseplate subunits having lysozyme activity, being similar to the gene products in T4-like phages   . In OANVAlong with the categories typical for dsDNA phage genomes, additional functions were also predicted. PANV1 gp36 was predicted to be a phospholipase  and thus is possibly involved in lipid metabolism. PANV1 gp37 putatively belongs to the NAD and ADP-ribose (NADAR) superfamily, having a hydrolyzer activity and taking part in carbohydrate derivative metabolic processes . PANV1 gp51 had matches to mechanosensitive channel proteins, which are involved in transmembrane transport . However, a transmembrane helix was predicted in this protein with only ~0.6 posterior probability by TMHMM v. 2.0. In PANV2, gp30 is a putative transpeptidase, which is involved in peptidoglycan cross-linking .Myoviridae of Type 2,” adopting the structural organization of the myophage T4 neck. PANV2 was assigned to “Siphoviridae of Type 1 cluster 5,” adopting the structural organization of the siphophage SPP1 neck. Both OANV1 and OANV2 were predicted to belong to “Podoviridae of Type 3,” adopting the structural organization of the podophage P22 neck.  and short noncontractile tails  (see 13 and 3.7 × 1013 PFU/mg of protein) using two negative stains and all demonstrated consistent particle morphology. The protein patterns of the purified OANV1 particle samples were identical to each other  (a) or Nano-W (b). Scale bar is 100 nm in a for a and b. (c) Polyacrylamide gel electrophoresis of purified virus samples which were prepared in parallel  and used in transmission electron microscopy in a and b, respectively. M, marker (PageRuler unstained protein ladder). Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Caudovirales phage genome assembly (GenBank accession number LR798304), which was obtained from metagenomes from the Římov Reservoir (freshwater human-made pond), Czech Republic  between PANV1 and PANV2 and the highest (~52%) between OANV1 and OANV2. When the complete sequences were used as queries in blastn searches  against all virus sequences (NCBI taxonomy identifier [ID] 10239) in the nr nucleotide collection, typically only less than 3% of the whole-genome sequences could be aligned with other virus sequences available, highlighting the overall uniqueness of these Antarctic sea ice virus isolates. One exception was the match of the OANV2 genome to the uncultured Republic . The ove10.1128/mbio.00651-22.8FIG S3Caudovirales phage (GenBank accession number LR798304.1) found with blastx search against nr protein database (a) and scaffolds found with blastn search against IMG/VR database (b). A full list of scaffolds is presented in FIG S3, PDF file, 1.5 MB.OANV2 and the selection of similar virus genome sequences, as follows: uncultured Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.2TABLE S2Table S2, PDF file, 0.1 MB.Putative functions assigned to PANV2 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.3TABLE S3Table S3, PDF file, 0.2 MB.Putative functions assigned to OANV1 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.4TABLE S4Table S4, PDF file, 0.1 MB.Putative functions assigned to OANV2 ORF products. Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.5TABLE S5Table S5, PDF file, 0.1 MB.Scaffolds with several (at least three) regions recruited as hits in the blast search against IMG/VR with Antarctic virus isolates whole genomes as queries (dated 9 December 2020). Download Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In contrast, a similar blastn search with PANV1, PANV2, OANV1, and OANV2 whole-genome sequences as queries against the Integrated Microbial Genomes/Virus (IMG/VR)  databasePANV2 was similar to scaffolds assembled from saline water microbial communities from Ace Lake, Antarctica , and one10.1128/mbio.00651-22.9FIG S4FIG S4, PDF file, 0.2 MB.PANV2 and the selection of similar scaffolds found with blastn search against IMG/VR database. A full list of scaffolds is presented in Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.00651-22.10FIG S5FIG S5, PDF file, 0.1 MB.OANV1 and the selection of similar scaffolds found with blastn search against IMG/VR database. A full list of scaffolds is presented in Copyright © 2022 Demina et al.2022Demina et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the A blast search of PANV1, PANV2, OANV1, and OANV2 genome sequences against the IMG/VR spacer databases did not yield any information about potential additional hosts of these viruses. No reliable hits were detected to CRISPR spacers from isolate genome, and the hits to metagenome-derived spacers were on short metagenome contigs that could not be affiliated taxonomically.Shewanella or Flavobacterium sp.     and Pseuctively) . In the low zero . Phage alow zero , 61.Caudoviricetes. A few PANV1 proteins may take part in lipid and carbohydrate metabolism and membrane transport, thus possibly being auxiliary metabolic genes, which is seen commonly in marine phages   or Tris-HCl (pH 7.5 and 9). After all incubations, virus infectivity was assessed by plaque assay as described previously  when two groups were compared. Groups were considered statistically not different if the P value was >0.05.Viruses and bacteria  were groM MgSO4) , SM buffeviously . A singl550 of ~0.8) were infected with a multiplicity of infection (MOI) of ~0.001 and incubated aerobically at 4°C. Samples in which cells were replaced with broth were used as controls. To determine the number of unbound viruses, samples were diluted in 4°C broth (1:10 or 1:100), cells were removed , and supernatants were subjected to plaque assay. The percentage of adsorption was calculated from all (particles in broth) and unbound particles (in infected cultures) as follows: % bound particles = [/all] × 100%. Adsorption rate constant was calculated as (k) = [2.3/(B × t)] × log(p0/p), where B is cell concentration, p0 and p are free virus concentrations at time point zero and after time period t, respectively  was infected with PANV1 or PANV2 (MOI of 10) and incubated aerobically at 5°C. Uninfected culture was used as a control. The numbers of infective free viruses in culture supernatants  were determined by plaque assay. IceBac 419 and IceBac 430 cells (OD550 of ~0.8) were collected  and resuspended in OANV1 or OANV2 virus stocks, respectively, (MOI of ~8) or in broth (uninfected controls). During the growth, the numbers of free viruses and viable cells in supernatant and pellet fractions  were determined by plaque assay.For life cycle studies, IceBac 372 , and assembled with SPAdes v. 3.9.0 .https://www.geneious.com) was used for sequence handling. ORFs were predicted using Glimmer, GeneMarkS , MetaGeneAnnotator (http://metagene.nig.ac.jp/), and FGENESV (http://www.softberry.com/berry.phtml?topic=virus&group=programs&subgroup=gfindv). GC content was calculated with Genomics %G~C Content Calculator . Transmembrane helices were predicted using TMHMM server v. 2.0 . Whole-genome comparisons for overall nucleotide identity were done with EMBOSS stretcher  database  using thThe genome sequences of PANV1, PANV2, OANV1, and OANV2 were compared to the IMG/VR spacer databases (both “isolate” and “metagenome”)  using blSequences are available in the GenBank database .


Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus–host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains. Foot-and-mouth disease virus , the causative agent of the eponymous disease, is a rapidly evolving pathogen that impacts livestock across much of the world. Currently, there are six antigenically distinct serotypes in circulation defined by the VP1 capsid-encoding region of the genome: A, O, Asia1, SAT 1, SAT 2, and SAT 3. Multiple serotypes regularly co-circulate in endemic regions and animals have been found to be naturally coinfected with multiple serotypes . FurtherThe clinical phase of FMDV infection in naïve cattle is characterized by fever and vesiculo-erosive lesions in the mouth and on the feet . ImportaHistorically, laboratory investigations into FMDV recombination detected genomic crosses of conditionally lethal mutants in tissue culture ,15,16,17The mechanism by which picornavirus recombination is believed to take place is termed ‘copy choice’ or ‘template switching’, and consists of the RNA-dependent RNA polymerase (RdRp) switching from one template genome to another during replication, thereby creating a hybrid product ,24. WhilThe present work builds upon the recently-published investigation that followed the clinical progression and infection dynamics of cattle coinfected with FMDV serotypes A and O . Within Virus samples used for the current investigation were derived from a set of experimental studies of FMDV coinfection in cattle . SpecifiOropharyngeal fluid sampling was paused during the acute phase of infection to not interfere with disease pathogenesis. At 10 or 14 days after the serotype O superinfection, sampling of OPF was performed at 3–4 day intervals over the course of 14–28 days , Table 1In order to investigate the virus populations associated with the emergence of interserotypic recombinant FMDV, all VI-positive OPF samples isolated following superinfection with FMDV serotype O were Illumina-sequenced as previously described . All samA)/10 days post-infection with FMDV-O (dpiO) or 35 dpiA/14 dpiO was also the first observation of recombinant viruses for all animals in which recombinants were detected. Importantly, these timepoints fell within the early (neoteric) phase of the second FMDV infection, when transmission remains viable [A/17 dpiO through study close at 56 dpiA/35 dpiO  FMDV-A, non-recombinant  FMDV-O, and/or interserotypic recombinant viruses , Table 1s viable . Further/35 dpiO . The samx-axis representing coding region 2A–3D, which is homologous  between both parental serotypes (A/10 dpiO and 35 dpiA/14 dpiO) included multiple interserotypic recombinants. Minority recombinants identified commonly became dominant in the following OPF samples. For example, a minority recombinant virus in the #1929 52 dpiA/31 dpiO sample was identified as the majority recombinant virus in the subsequent 56 dpiA/35 dpiO sample  suggested that sequence homology between the parent genomes was important in recombination. RDP4 software version 4.101 [p < 0.0001) suggested that some of the breakpoints identified in the current study may be classified as hotspots.The locations of interserotypic recombinant breakpoints were analyzed. The mechanism of recombination is thought to be enabled by the RdRp (encoded by 3D) switching templates due to high regional homology between the donor and acceptor templates . In ordeon 4.101 ,30 was uon 4.101 . BreakpoA/24 dpiO–56 dpiA/45 dpiO) provided an indication of the genomic change detected over the course of the study (A) to the FMDV-A-derived segments of each animal’s final VI-positive OPF (45 dpiA/24 dpiO–56 dpiA/45 dpiO) provided an indication of the genomic change that took place after coinfection  during the period of coinfection than over the entire study period (The average number of substitutions/nucleotide/day (s/n/d) in cattle with persistent serotype A infections that had no evidence of recombinant viruses (#1931 and #1939) were 1.04 × 10 regions . In catt−5 s/n/d . For boty period . HoweverChanges in protein coding that took place over time were analyzed in viral genomes obtained from serial OPF samples. Specifically, this analysis focused on instances in which one virus replaced another as the dominant species. In comparing the FMDV-O inoculum to the FMDV-O-derived segments of succeeding recombinants, several nonsynonymous changes were identified in structural proteins . Most noA/14 dpiO #1914 sample included a dominant (>99%) interserotypic recombinant composed of an FMDV-O genome from the 5′UTR through a portion of the 2C coding region and an FMDV-A-derived genome thereafter  was 2.8 mm2. This was substantially larger than the average for the FMDV-A inoculum, 0.6 mm2, and smaller than FMDV-O virus plaques, 4.4 mm2. Interestingly, the 49 dpiA/28 dpiO plaques (FMDV-A at study end) averaged 1.5 mm2, more than twice the size of the FMDV-A inoculum plaque average.Samples were serially diluted and incubated for 24 h on LFBK-αvβ6 cells . Plaques260 peak absorbances measured per fraction. Specific infectivity measures, defined as the ratio of viral particles to plaque forming units (VP/PFU) were 7.94 × 104 for the FMDV-A inoculum, 2.10 × 105 for the FMDV-O inoculum, 5.86 × 104 for the 35 dpiA/14 dpiO #1914 recombinant, and 1.41 × 105 for the 49 dpiA/28 dpiO #1914 non-recombinant FMDV-A virus  of persistently infected cattle that were superinfected with a heterologous virus strain. By contrast, within the same studies, recombinant viruses were not detected in any samples from vesicle lesions . Given t% of persVirus populations present in OPF samples were found to be highly diverse. This was particularly true of those sampled relatively early following FMDV-O superinfection . Those sConsistent with the complexity of OPF virus populations, substitution rates were higher during the periods of superinfection compared to the periods of single virus infection. This elevated rate of within-host evolution during FMDV co-infection suggests that distinct immune mechanisms and/or selective pressures may exist during co-infection when compared to single strain infection.Findings from in vitro assays did not indicate significant growth differences between the parental viruses and a selected interserotypic recombinant. The specific infectivity of this recombinant had a lower ratio of viral particles to plaque forming units than its parents or its successor. That specific measurement is impacted by multiple aspects of the virus infection cycle, and thus does not necessarily indicate that the recombinant is more fit overall. Nevertheless, that finding suggests that packaging an interserotypic recombinant FMDV genome into an FMDV-O capsid does not incur a fitness loss. This is consistent with a recent investigation of cell culture coinfection which demonstrated trans-encapsidation of serotypes FMDV-Asia 1 and FMDV-O genomes in heterologous capsids .All recombinant viruses detected in these studies encoded FMDV-O capsids. Serotypes A and O are antigenically distinct, meaning that acquired immunity against one does not protect against infection with the other serotype. Thus, the animals’ capacity to neutralize FMDV-O capsids is presumed to have been weaker directly following superinfection with that serotype, while the adaptive response to FMDV-A capsids was already well-established at that time . This coThere was no consistency in the dominance of viruses detected in final OPF samples across the 12 superinfected cattle. Amino acid analysis did not indicate any specific change related to dominance of one genome over another. Nevertheless, the success of interserotypic recombinant viruses over non-recombinant FMDV-O viruses is curious. This is because both groups encode FMDV-O capsids, presumably subject to comparable immune pressure. Analysis of nonsynonymous substitutions identified several amino acid changes in recombinants, most notably a change at the receptor binding site. However, this change was present in the non-recombinant FMDV-O genomes as well; thus, it does not suggest an advantage that was unique to recombinants. A more substantial difference between the recombinants and the non-recombinant FMDV-O viruses are the FMDV-A-derived portions of the recombinant genomes. FMDV recombination hotspots have been consistently identified to fall between the functional units of the genome, e.g., capsid, proteases, polymerase etc. ,38. If tMechanistically, when the viral polymerase switches templates during a recombination event, it has been proposed that this movement is facilitated by elevated local sequence identity and related secondary structure elements between the donor and acceptor templates ,40,41,42The mechanisms by which FMDV maintains persistent infection are incompletely understood . StudiesIn all animals in which recombinants were detected, chimeric viruses were found to be the dominant virus in the first OPF sample obtained following FMDV-O infection, corresponding to 10–14 days after superinfection. Because recombinants were already dominant at these sampling time points, it is likely that these viruses were common, if not dominant, at earlier timepoints as well. However, the precise timing of onset of detection of recombination was not determined herein. Importantly, those earlier time points are when FMDV transmission is more likely during single-virus infections . This isThe samples used in the current study were derived from persistently infected carrier animals that were superinfected with a heterologous virus. These animals thereby represent a distinct category as they were both persistently and acutely infected, regardless of their clinical status. Coinfection is not uncommon in FMD-endemic regions where multiple serotypes are in circulation ,2,3,4 anWe have previously characterized the neoteric (early) subclinical phase of FMDV infection as distinct from the more commonly recognized persistent phase of infection based on higher quantities of virus detectable in oronasal secretions. This neoteric subclinical shedding of FMDV has an increased probability of transmission compared to persistently infected carriers that are not superinfected ,13,51. I6 TCID50 of FMDV A24, after which all animals developed clinical FMD of expected severity. After either 21  or 35  days, the animals were superinfected with FMDV O1 Manisa using the same titer and route. Details of the pathogenesis across these studies have been previously published [Virus samples used for the current investigation were derived from a previously published study investigating the pathogenesis of FMDV coinfection in cattle, using two bovine-derived and well-characterized FMDV isolates of strains A24 Cruzeiro and O1 Manisa . The 12 ublished . OropharDeep-sequencing of virus isolates analyzed in the present work was performed on the NextSeq 550 (Illumina) from OPF samples passaged once in LFBK-αvβ6 cells . FMDV fuCLC Genomics Workbench v. 20–v. 21 was used to filter reads for quality and length and competitively assemble them to each of the two reference genomes corresponding to the two inocula, A24 Cruzeiro (GenBank #AY593768) and O1 Manisa (#AY593823). The identification of recombinant genomes was initially based on inconsistencies in read mapping, but later confirmed by Sanger sequencing amplicons created with breakpoint-crossing primers, de novo assembly, resequencing, sequencing of un-passaged samples, chimeric read analysis and haplotype reconstruction; all methods covered in brief here are detailed in .Breakpoints were identified both at the consensus and subconsensus level through a combination of significant reference genome coverage changes, chimeric read identification and haplotype reconstruction. A subset of samples from each animal were also Sanger sequenced as noted above. Chimeric reads were mapped to the CDS of each reference genome and the site-wise coverage values were extracted. The product of site-wise chimeric read coverage between each reference was calculated such that putative chimeric reads that only mapped to one parental genome were discounted. This data set was then compared to breakpoints predicted among a large, 769 FMDV CDS data set (767 with the inoculum viruses removed) curated and published by Aiewsakun, Pamornchainavakul and Inchaisri  using thFMDV populations are comprised of numerous genomic variants, regularly called a mutant swarm or quasispecies. For FMDV in particular, variant virus genomes within FMDV populations have been found to fall into clades or haplogroups that can persist over time ,53. In oA/14 dpiO OPF sample from animal #1914, and the FMDV-A (non-recombinant) virus identified in the 49 dpiA/28 dpiO sample from animal #1914 were amplified on LFBK-αvβ6 cells for 24 h [Each inoculum, the recombinant virus identified in the 35 dpifor 24 h . Next, efor 24 h  in Imagefor 24 h .A/14 dpiO and 49 dpiA/28 dpiO OPFs from animal #1914. Each sample was serially diluted and plated on LFBK-αvβ6 cells in 6-well cell culture plates . Discrete plaques were picked, transferred to 500 μL PBS and stored at −70 °C. Thawed plaques were then incubated on LFBK-αvβ6 cells for 24–48 h (until cytopathic effect was observed) and stored at −70 °C. Homogeneity of each isolate was confirmed through targeted RT-PCR as well as Illumina sequencing as described above. Each virus was then amplified in LFBK-αvβ6 cells in 2 L roller bottles. Lastly, each virus was concentrated using polyethylene glycol precipitation and titrated.To ensure genetic homogeneity for subsequent analyses, plaques were isolated for each inoculum as well as both the 35 dpi2 TCID50/mL at 2 h. Specific infectivity was measured via sucrose gradient diffusion followed by spectrographic analysis [260 was measured for each virus within the same gradient fraction corresponding to the encapsidated virus absorbance peak.Growth rates of each virus were measured in LFBK-αvβ6 cells, with aliquots titrated at 1, 2, 4, 7, and 24 h. Growth was below the level of detection for 1 and 2 h timepoints except for FMDV-O which averaged 2.17 × 10analysis ,58; Samp