Implications of mixed viral infections on plant disease ecology and evolution.

Mixed infections of plant viruses are common in crops and represent a critical biotic factor with substantial epidemiological implications for plant viral diseases. Compared to single-virus infections, mixed infections arise from simultaneous or sequential infections, which can inevitably affect the ecology and evolution of the diseases. These infections can either exacerbate or ameliorate symptom severity, including virus-virus interactions within the same host that may influence a range of viral traits associated with disease emergence. This underscores the need for a more comprehensive understanding of how the order of virus arrival to the host can impact plant disease dynamics. From this perspective, we reviewed the current evidence regarding the impact of mixed infections within the framework of simultaneous and sequential infections in plants, considering the mode of viral transmission. We also examined how the temporal order of mixed infections could affect the dynamics of viral populations and present a case study of two aphid-transmitted viruses infecting melon plants, suggesting that the order of virus arrival significantly affects viral load and disease outcomes. Finally, we anticipate future research that reconciles molecular epidemiology and evolutionary ecology, underlining the importance of biotic interactions in shaping viral epidemiology and plant disease dynamics in agroecosystems.

In South Korea, the cultivation area of elephant garlic (Allium ampeloprasum) is increasing as elephant garlic is milder and sweeter than garlic (A. sativum) (Kim et al., 2019; Lu et al., 2011). Viral diseases can decrease garlic productivity by up to 50% in South Korea (Nam et al., 2002). In 2022-2023, virus-like symptoms such as mosaic and yellow stripes were observed on leaves of elephant garlic in a 432㎡farm with disease incidence of approximately 40% in Yangpyeong-gun, Gyeonggi-do, South Korea. Seventy-two leaf samples were randomly collected from symptomatic plants in 2022 (n=46) and 2023 (n=26). Total RNAs were isolated from individual samples using the Total RNA Prep Kit (BioFact, Daejeon, Korea), and then two-steps RT-PCR was performed using the First Strand cDNA Synthesis kit (Thermo Fisher Scientific) and the TaKaRa TaqTM (TaKaRa Bio Inc.). These samples were tested for 13 viruses with virus-specific coat protein primers including garlic common latent virus (GarCLV) (supplementary Table 1). In 2022, GarCLV, garlic virus (GarV)-B, GarV-C, and GarV-D were detected with the expected amplicon sizes of their CP genes (960, 735, 780, and 753 bp, respectively) in four different plants. In 2023, the CP gene of GarCLV was detected in 26 samples and 4 of 26 samples were positive for GarV-B. The leaves infected with GarCLV and GarV-B in mixed infection showed synergistic effect with extended mosaic and yellow stripes than the leaves with single infection (supplementary Fig. 1). All amplicons were cloned into a pGEM-T Easy vector (Promega Co., USA), and sequenced at Bionics Co. Ltd., South Korea. The resulting nucleotide (nt) and amino acid (aa) sequences were analyzed using DNAMAN software version 5.1. Since all isolates were collected from a farm in Yangpyeong-gun, name of these isolates started with "YPG." The nt and aa sequences of the isolates were compared with those of other strains/isolates. All 27 GarCLV-YPG isolates sequences were deposited (Accessions: OP981636, and PP533185-PP533210). The GarCLV-YPG sequences shared 78.90%-94.40% nt and 92.10%-99.40% aa identities with other GarCLV strains and isolates, and they showed higher similarity (99.40% aa) to isolates produced from A. sativum in China and India (supplementary Table 2). GarV-C-YPG showed the highest similarity (99.20% aa) to isolate G81(GenBank MN059141) from A. sativum in China. GarV-D-YPG showed the highest similarity (99.20% aa) to isolates (G82, GenBank MN059388; BR, MT279193) from A. sativum in China and Brazil. Twenty-two quinoa plants (Chenopodium quinoa, local lesion host) were individually inoculated using the sap from 22 GarCLV infected plants. Chlorotic and necrotic spots appeared on inoculated leaves 12 days post-inoculation; no chlorotic and necrotic spots symptoms were observed on any other leaves except for the inoculated leaves. RT-PCR was performed and the targeted amplicon size for GarCLV was detected. In transmission electron microscope, filamentous particles of approximately 620-730 nm length and 12 nm diameter, similar to the particle description for members of the family Betaflexiviridae, were observed in the saps of symptomatic leaves of elephant garlic and quinoa plants infected with only GarCLV. To the best of our knowledge, this is the first report on GarCLV detection in elephant garlic in South Korea. We hypothesized that the presence of GarCLV in mixed infection with GarV-B might have increase the symptom severity in the elephant garlic. Further study is needed to proof the synergistic effect in mixed virus infection.

BackgroundPlant-infecting viruses cause severe crop losses throughout the world. The frequent occurrence of mixed infection (co-existence of multiple viruses in a single infected plant) in the field makes the situation more alarming. Mixed infection of two viruses often boosts the fitness of either of the viruses and thus increases disease severity. Maize lethal necrosis disease and rice tungro disease are some devastating examples. This study focused on the mixed viral infection between begomovirus, a ssDNA virus, and potato virus X (PVX), a ssRNA virus.ResultsV2 protein encoded by a monopartite begomovirus, pepper leaf curl Bangladesh virus (PepLCBV), promotes the accumulation of PVX. When Nicotiana benthamiana plants were infected with both PVX and PepLCBV, aggravated PVX-induced symptoms and increased PVX titre were detected compared to plants infected with PVX alone. This observation suggests that PepLCBV promotes the viral titre of PVX during mixed infection. Next, when PepLCBVΔV2 infectious clone was co-infiltrated with PVX, no increase in PVX-associated symptoms was observed in the plants, which confirmed that the V2 protein plays a pivotal role in mediating the interaction between these two viruses. PepLCBV-V2 was experimentally identified as a potent suppressor of post-transcriptional gene silencing. Its interaction with suppressor of gene silencing 3 (NbSGS3) was confirmed through yeast two-hybrid and BiFC. Silencing of NbSGS3 facilitated PVX pathogenesis. Next, specific amino acids responsible for the suppressor activity of the V2 protein were identified, and the silencing suppressor mutant V2 failed to boost the PVX titre. A similar observation was recorded when the association of PVX with a bipartite begomovirus, tomato leaf curl New Delhi virus, was checked in both N. benthamiana and tomato.ConclusionThe present study, for the first time, reports that begomoviruses promote the pathogenesis of potato virus X during mixed infection and identifies begomoviral pre-coat protein (V2/AV2) as a key player in mediating this virus-virus interaction. We further demonstrated that the silencing suppression activity of V2 is important for regulating PVX titre in mixed-infected plants.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12985-025-03005-0.

Effect of mixed infections of Sindbis and La Crosse viruses on replication of each virus in vitro

Mixed Viral Infection Identified Using Heteroduplex Mobility Analysis (HMA)

Mealybug wilt of pineapple (MWP) is a major viral disease of pineapple (Ananas comosus) worldwide, including Ghana (Sether & Hu, 2002; Sarpong et al., 3). In Ghana, MWP causes yield loss estimated at about US$248 per hectare (Sarpong et al., 3). Recently, Nyarko & Asare-Bediako (2) detected Pineapple mealybug wilt associated virus-1 (PMWaV-1) and PMWaV-3 (genus Ampleovirus) from 9 of 24 MWP-symptomatic pineapple plants from Ghana. However MWP is also associated with three additional ampeloviruses PMWaV-2, PMWaV-4 and PMWaV-5 (Gambley et al., 1) and further testing was done to determine if these viruses contributed to the disease in Ghana. Nucleic acid extracts of the same 24 pineapple plants (Nyarko & Asare-Bediako, 2), which had typical MWP symptoms, including tip dieback, downward curling, reddening, and wilting of the leaves and in which PMWaV-1or PMWaV-3 were detected, were tested using RT-PCR with the 223/224 primer pair which targets the HSP70h gene region of PMWaV-2 (Sether et al., 5). PMWaV-2 was detected in 15 of the samples (62.5%), suggesting its abundance in pineapple fields in Ghana. Five amplicons corresponding to the expected size (610 bp) were purified and directly sequenced in both directions. Sequence comparison using BLASTn showed that all the Ghanaian PMWaV-2 isolates (GenBank Accession Nos. MN427642-MN427645) shared 98.9-100% nucleotide identity with each other and 98.2-100% nucleotide identity with sequences of isolates in GenBank (e.g. KT322167, HE583226 and FN825676). To the best of our knowledge, this is the first report of the presence of the PMWaV-2 in Ghana, as well as in Africa. So far, this work and that of Nyarko & Asare-Bediako (2) has shown the presence of PMWaV-1, PMWaV-2 and PMWaV-3 in Ghanaian pineapple. Sixteen out of the 24 samples (66.7%) tested were infected with at least one of the three species of ampeloviruses identified. PMWaV-2 had the highest infection rate (62.5%), followed by PMWaV-1 and PMWaV-3 with infection rates of 33.3% and 8.3% respectively. Mixed viral infections were detected in seven of the sixteen PMWaV-infected pineapple samples, where five were co-infected by PMWaV-1 and PMWaV-2 and the other two were co-infected by PMWaV-1, PMWaV-2 and PMWaV-3. This has serious implications for the epidemiology and management of MWP. Mixed infections can result in recombination of viral species leading to the generation of variants showing novel genetic features, which may cause severe damage to crops (Syller, 6). MWP has been reported to be highly prevalent in pineapple fields in Ghana (Sarpong et al., 2017). There is therefore the need to identify an effective strategy to manage the disease in order to ensure the sustainability of the pineapple industry in Ghana.

Bell pepper ( Capsicum annuum L.), a crop of global importance, is threatened by mixed viral infections, such as Potyvirus yituburosum (PVY) and Tobamovirus fructirugosum (ToBRFV). This study evaluated the antiviral potential of green-synthesized silver (Ag), silicon dioxide (SiO 2 ), and cerium dioxide (CeO 2 ) nanoparticles (NPs) against these viruses in seeds and seedlings. NPs were synthesized using aqueous walnut shell extracts. The nanoparticles were characterized by EDX and TEM. In two experimental phases, bell pepper seeds were treated with different concentrations of nanoparticles (50–1000 mg L −1 ) and subsequently exposed to PVY, ToBRFV, and both. Viral transmission was assessed by qELISA in seeds and seedlings. Seedling development, germination rates, and virus incidence were measured; phytotoxicity (CF 50 ), antiviral efficacy (IC 50 /IC 90 ), and the selectivity index (SI) were calculated. The results showed a significant reduction in viral load and inhibition of PVY and ToBRFV transmission in both single and mixed infections, especially at doses ≥400 mg L −1 . CeO 2 nanoparticles exhibited the highest selectivity indices (SI > 3000) and no phytotoxicity. Ag and SiO 2 nanoparticles also exhibited antiviral activity, but with slightly higher CF 50 values. In the greenhouse, seedlings grown from seeds treated with nanoparticles showed a reduction in viral symptoms and an increase in SPAD index and height. In conclusion, bio-nanoparticles, especially CeO 2 , are promising antiviral agents against PVY and ToBRFV in pepper cultivation. Their high sensitivity and low toxicity make them good candidates for the sustainable management of plant viruses, justifying further field-scale evaluation.

Objective : to identify current clinical and laboratory characteristics of infectious mononucleosis in children. Materials and methods : this study presents an analysis of 50 clinical cases of infectious mononucleosis in children hospitalized at the Orenburg Regional Clinical Infectious Diseases Hospital in 2025. To confirm the diagnosis, blood and saliva were tested using polymerase chain reaction to detect Herpes virus DNA, nasopharyngeal swabs were tested using PCR for respiratory viruses and SARS-CoV-2, and oropharyngeal mucus was bacteriologically tested for opportunistic pathogens. Results : it was established that infectious mononucleosis was characterized by the development of typical syndromes (intoxication, lymphoproliferative, hepatosplenic). However, at the onset of the disease, intoxication syndrome and oropharyngeal changes predominated, often leading to misdiagnosis and lack of appropriate etiotropic treatment at the prehospital stage (100%) and late hospitalization of children (66%). Conclusions : the overwhelming majority (78%) of patients had liver damage with the development of cytolysis syndrome. More than half (56%) of children had mixed infections: viral-viral (26%) and viral-bacterial (30%), which aggravated the severity of infectious mononucleosis and required complex etiotropic therapy. A comparative analysis of the course of infectious mononucleosis in children with mono- and mixed infections revealed significant differences in the severity of cytolysis syndrome in children with mixed viral infections with long-term recovery of AST and ALT levels (14-28 days), which must be taken into account in the rehabilitation program for such patients.

Objective: Many intractable diseases, like Multiple Sclerosis (MS), Amyotrophic Lateral Sclerosis (ALS), Diabetes Mellitus Juvenilis and Endometriosis are treated today without knowing the cause and true nature of the diseases. Using the Bi-Digital O-Ring Test (BDORT) as a diagnostic method we are able to find out cause and nature of some diseases. Materials and Methods: The patients with different intractable diseases were analyzed in our prospective studies using BDORT. Ten patients with MS, 2 with ALS, 8 with Diabetes mellitus Juvenilis and their mothers, and 29 patients with Endometriosis were investigated, based on the original work by Prof. Omura, Y. Results: According to BDORT, in all cases MS and ALS the infection of brain, in the left or right part (parieto-occipital) corresponding to sensory-motor cortex was found. Those were mixed infections, and same infections were in genital and urinary organs of each patient. Deposits of mercury indicated that the primary infection was in genital and urinary organs. All patients had Chlamydia Trachomatis as a main infection, and mixed viral and bacterial infections. Eleven patients had Candida Albicans too. Patients with Diabetes Mellitus had infections of endocrine pancreas with Cytomegalovirus as a main infection and Candida Albicans and mixed bacterial infections. Their mothers had also CMV infections of uterus, IgG antibody of CMV was confirmed in their mothers but not in children. All patients with Endometriosis had Herpes virus 6 infection of endometriosis area. We tested 7 patients with Elisa method and all of them had high level of IgG antibodies of HSV 6. Conculsion: These findings obtained by BDORT indicate that early diagnosis may be made of MS, ALS, Diabetes Mellitus Juvenilis and Endometriosis using BDORT. Having that in mind, may be prevent or treat these patients, with the view to cure cause and not the symptoms. According to these findings MS and ALS are Sexual Transmisive Diseases which can be prevented as other STD. Diabetes Mellitus Juvenilis is a conatal disease, which can be prevented by inspecting the mothers by BDORT before conceptions or inspecting the newborns by BDORT and treating the CMV infections before destroying beta cells.

Background:Hydatid cyst is a disease that is transmitted from the lactating animals that come into contactwith a person, and its danger is to generate hydatid cysts that may put pressure on some vital organs inthe human body. Here comes the role of joint infection with other pathogens such as the hepatitis C virus,especially in the same organs that are infected with the parasite, which may result from these mixed infectionsserious disease complicationsAim of study: The present study aimed to examine the possibility of a relationship between interleukin-8concentration level with the size of Hydatid cyst patients with mixed infection of hydatidosis and hepatitisC virus.Patients and Methods: The current research was run for the period January 2019 to January 2020. Thepatients were referred to AL-Furat al-awsat Hospital , Al-Sadder medical city, Public Health Laboratoryand Al-Hakeem Hospital in Al- Najaf governorate, Iraq. Eighty samples were divided into two groups. Thisfirst group was comprised of 40 patients with hydatidosis, the second group comprised of 40 patients withhydatidosis and hepatitis c virus mixed infection. The patients were aged 10 to 60 years old.Result: A higher interleukin-8 concentration was observed in patients with hydatidosis compared to thehealthy group, as the interleukin-8 concentration was (89.36) While the concentration of interleukin- 8reached in the healthy group (30 .14) ,while A high concentration of interleukin 8 is observed in combinedinfection of hydatidosis and hepatitis C virus compaer with healthy group , As the concentration of interleukin8 in the combined infection of hydatidosis and hepatitis C virus (108.73) while in the healthy group (30.14 ).Conclusion: The presence of a high level of interleukin-8 in mixed-infection patients is higher than insingle-infection patients, which may lead to exacerbation and complications of hydatidosis disease.

Introduction. Respiratory viruses (RV) circulate throughout the world and in all seasons of the year. Long-term monitoring of the distribution of respiratory pathogens is necessary to analyze the relevance of diagnostic systems to current viral isolates, to assess the risks of infection and the need for vaccine development and use, as well as to investigate the interdependence of RV reproduction in mixed infections. Objective — to study the causative agents of acute respiratory viral infections (ARVI) in Moscow during 2011–2022 by reverse transcription with subsequent polymerase chain reaction with fluorescent hydrolysis probes detection in real-time (RT2-PCR). Materials and Methods. Nasopharyngeal swabs from 3908 patients with acute respiratory infections were examined by the RT2-PCR. Results. Monitoring of RV spread in Moscow showed cyclical changes in frequencies with three dominant species: influenza A virus (up to 31.3%), respiratory syncytial virus (up to 24.8%) and human rhinoviruses (up to 21.3%) in 2011–2020. The increase in the portion of unidentified clinical specimens from 1.2 to 28.5% in 2022 indicated incomplete accordance of diagnostic systems to modern RV isolates or the emergence of new species or strains of pathogens. Unidirectional changes in dynamics were registered for 5 out of 9 studied RVs with correlation coefficients of 0.43–0.79. High frequencies of mixed acute respiratory viral infections (up to 33.4%) along with unidentified samples do not allow us to accurately assess the risks of infection with various RV in Moscow, but prove the necessity of preventing infectious diseases with the most common RV. Conclusion. Analysis of the dynamics of RV frequencies in Moscow showed the preservation of the dominant species: influenza A virus, respiratory syncytial virus and human rhinoviruses. During the period of vaccination against COVID-19, the proportion of seasonal coronaviruses increased.

Ultrastructure of mixed plant virus infection: Bean yellow mosaic virus with cowpea severe mosaic virus or cowpea mosaic virus in bean

Scientific relevance. Plant viruses cause a significant economic loss to potato production, especially if plants are infected at early growth stages and infections are mixed. Viral diseases reduce both yield and quality of harvested crops. Detection and identification of plant viruses are key important to prevent their spreading and to achieve potential yield predetermined by characteristics of varieties.Research methods. Seven potato varieties, bred in Russia and overseas, were used in the field experiment: Smak, Avgustin, Yantar, Laperla, Labella, Red Lady, Sante, Belmonda. Viral infection rate was measured by the percent of plants with symptoms to the total number of plants. In addition to infection frequency, a disease rate was described after visual estimation. Total RNA was isolated from the collected leaves according to Bekesiova I. et al. 1999 [13]. Qualitative and quantitative estimation of plant viruses in the samples were conducted by single-step real-time RT-PCR with fluorescent detection with the Applied Biosystems QuantStudio 5 and commercial kits “Potato Virus X, Y, M, L, S, A, PSTVd-RT” (Syntol Company) according to the official protocol of the kits.Results. As a result of our research, symptoms of mixed viral infection were described for potato varieties depending on concentrations and proportions of these viruses in a plant. Mixed viral infection in the potato field in Primorsky Krai comprised PVY, PVX, PVA, PVS, PVM, also PLRV and PSTVd.

Dominance of Cold-Adapted Influenza A Virus over Wild-Type Viruses Is at the Level of RNA Synthesis

We report the construction and characterization of deletion mutants in the herpes simplex virus type 1 gene encoding the immediate-early protein ICP0. In the event that ICP0 proved to play an essential role in virus replication, ICP0-transformed Vero cells were generated to serve as permissive hosts for such mutants. Two mutants, dlX0.7 and dlX3.1, were isolated in these cells by a marker rescue-transfer procedure involving the rescue of an ICP4 deletion mutant and the simultaneous insertion of a linked deletion in the ICP0 gene. Mutant dlX0.7 contained a 700-base-pair deletion in both copies of ICP0. The deletion lay entirely within the transcript specified by the gene. dlX0.7 induced the synthesis of an ICP0-specific mRNA that was approximately 0.7 kilobases smaller than the corresponding mRNA specified by wild-type virus. The 3.1-kilobase deletion in both copies of the ICP0 gene in mutant dlX3.1 removed the majority of the transcriptional-regulatory signals and coding sequences, retaining only sequences at the 3' end of the gene. As expected, no ICP0-specific mRNA was detected in dlX3.1-infected Nero cells (G418-resistant Vero cells). Both mutants grew in all cells tested, although their burst sizes were 10- to 100-fold lower than that of wild-type virus. Although the plaque sizes of dlX0.7 and dlX3.1 were equally small on Nero and ICP0-transformed cells, the plating efficiency of the mutants was 15- to 50-fold greater on ICP0-transformed cells than on Nero cells. The mutants exhibited modest interference with the growth of wild-type virus in mixed infections, an effect that was abolished by UV irradiation of the mutants, implying that interference required viral gene expression. Polypeptide profiles generated by the mutants in Nero cells were qualitatively similar to that of wild-type virus. Quantitatively, only slight reductions in the levels of certain late viral polypeptides were observed, a phenomenon also borne out by analysis of viral glycoproteins. Both mutants induced the synthesis of significant, although reduced, levels of viral DNA relative to wild-type virus. Taken together, the results demonstrate that ICP0 is not essential for productive infection in cell culture but that this protein plays a significant role in viral growth, as indicated by the impaired abilities of the mutants to replicate.