CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system  to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses. CRISPR/Cas13d coupled with an endogenous tRNA-processing system can be used to engineer resistance to multiple RNA viruses in potato. Diverse approaches of controlling RNA viruses in plants have been attempted including conventional breeding, genetic engineering and RNA interference4. However, due to the high rate of mutation and recombination of viruses, the virus-resistant plants could be overcome in a few years5. In addition, many plant viruses evolve and often develop various counter-defense mechanisms, such as viral suppressors of RNA silencing, nullifying the current antiviral approaches7.Plant viruses are obligate intracellular parasites replicating themselves by using the molecular machinery of their hosts. Plant viral diseases can cause severe yield loss and continuously threaten crop production worldwideSolanum tuberosum L.) is the world’s most important non-grain crop8 which is also sensitive to various biotic and abiotic stresses. Viruses are among the most significant biotic constraints in potato production. Potato is clonally propagated by planting tubers, further increasing the risk of virus infection. Until now, more than 50 different viruses have been reported to be able to infect potatoes worldwide. Among them, potato leaf roll virus (PLRV), potato virus Y (PVY), potato virus X (PVX) and potato virus S (PVS) have been recognized as the major potato viruses9. These four RNA viruses are all positive-sense single-stranded RNA (+ssRNA). PLRV is a type species of the genus Polerovirus, belonging to the family Solemoviridae11. An intriguing feature of PLRV is the phloem restriction. It can be delivered into phloem tissues by aphids, agro-inoculation or grafting, while not be transmitted by mechanical inoculation. Besides, PVY is a member of the genus Potyvirus in the family Potyviridae and is transmitted by aphids in a non-persistent manner or through potato tubers12. PVY is prone to high mutation and recombination rates, existing as a complex of strain groups, or variants14. On the other hand, PVS belongs to genus Carlavirus in the family Flexiviridae16. PVS can be transmitted by plant contact or several aphid species in a non-persistent manner17. Apart from this, PVX is a type species of the genus Potexvirus in the family Alphaflexiviridae18. The symptoms induced by PVY or PLRV are visible when infected with single virus, such as mosaic, mottled and crinkled leaves as well as leaf and vein necrosis in PVY infection19, and leaf rolling and stunting in PLRV infection10. Upon infection with a specific virus, PVY and PLRV are among the most damaging viruses of potato worldwide21. However, in the field, potato is usually challenged by multiple species of RNA viruses, which result in more significant loss of tuber quality and quantity22. For example, PVX or PVS causes varying degrees of tuber yield losses in the single infection, while the symptom and production loss can be more severe in the mixed infection23.Potato (26. The Cas9 and Cas12 of Class II were employed to combat DNA viruses in eukaryotes28. The type VI-A effector Cas13a (previously known as C2c2) has been characterized for cleaving single-stranded RNA29 and designed to interfere with viral RNA replication against the turnip mosaic virus via transient transformation assays30. Consistent with this, the CRISPR/Cas13a system can be engineered to target PVY genome and confer resistance to PVY in the transgenic potato plants by the stable transformation31. Similarly, CRISPR/Cas13a system could enable the plant to acquire potent defense against viral infection in both tobacco and rice plants32.The CRISPR/Cas system  is emerging as a promising tool for gene editing and sequence-specific regulation of gene expressionNicotiana benthamiana33, making it suitable for diverse RNA viruses targeting or detection34.Recently, a new Cas13 subtype, type VI-D (Cas13d), has been identified. Cas13d effector is relatively smaller compared to previously reported subtypes. Moreover, Cas13d was shown to have more robust RNA virus interference applications compared to LwaCas13a and PspCas13b variants in 35. To produce multi-virus-resistant plants, we sought to develop a robust multiple RNA-targeting CRISPR/Cas13d system conferring resistance to different RNA viruses. We show that CRISPR/Cas13d assembled with a PTG gene can reduce the multiplexed viral accumulation in transgenic potato plants, providing a new strategy for conferring broad-spectrum resistance to viral diseases in crops.It has been demonstrated that numerous gRNAs can be generated by employing the endogenous tRNA-processing system of plant cells, enabling the simultaneous targeting of multiple genomic lociCas13d gene (fusion with a 3 × HA tag sequence) from Ruminococcus flavefaciens XPD3002 (CasRx) and a polycistronic tRNA-gRNA (PTG) gene for simultaneous production of four gRNAs . The expression of Cas13d and PTG genes is driven by the UBQ10 (Arabidopsis ubiquitin-10) and AtU6 promoters, respectively  harboring a synthetic ely Fig. . Each sgAgrobacterium-mediated transformation. Transgenic lines were selected on kanamycin-containing plant regeneration medium. Over 15 independent transgenic lines were obtained. The expression of Cas13d was determined by western blotting  with relatively higher transgene expression were chosen for further experiments. Furthermore, phenotypes of transgenic lines growing on synthetic medium and soil were screened. We found that the transgenic plants expressing Cas13d/PTG were indistinguishable from wild-type plants under mixotrophic and autotrophic growth conditions  and wild-type (St-wt) plants were challenged with PLRV or PVYO. Accumulation of PLRV or PVYO was assessed at 15 and 25 days post-inoculation (dpi) by enzyme-linked immunosorbent assays (ELISA) and qRT-PCR assays, respectively. Resistance of St-PTG transgenic plants to PLRV or PVYO was evidenced by a strongly reduced accumulation of PLRV and PVYO in the systemic leaves  plants were exposed to PVS or PVX and the virus accumulation was determined at 15 and 20 dpi by qRT-PCR. We showed that, compared to wild-type control plants, Cas13d/PTG expression strongly inhibited PVS or PVX accumulation in the systemic leaves of the virus-infected transgenic plants  and wild-type (St-wt) plants. A PVY-resistant line (St-CP#7) expressing a single guide RNA targeting the CP gene of PVY31  can only reduce the accumulation and disease symptoms of PVY, but not PVX  failed to attenuate the accumulation of mixed viruses  can only reduce the accumulation of PVY  commonly occurred with diverse combinations22. For example, PVX, PVY and PVS are mostly found to make interactions, the percentage of PVX and PVY co-infection was equivalent with PVY and PVS co-infection in potato plants40. Our results indicated that the expression of four gRNAs (St-PTG) exhibited high interference with two or three mixed viruses. While interfere with the PVY accumulation when infected with PVY alone  has no effect on reduction of PVY accumulation in mixed infection  by employing an endogenous tRNA-processing system in rice protoplasts to efficiently guide Cas9 to multiple chromosomal targets45. These strategies would allow the expression of multiple gRNAs from a single transcript.To engineer multi-resistance to RNA viruses, multiplexed gRNA expression would be crucial. Therefore, the use of PTG would be necessary for engineering to resistance to multiple viruses. Except for the endogenous tRNA system used in this study, exogenous co-expression of Csy4 or self-cleaving ribozymes such as Hammerhead or HDV ribozymes flanking by each gRNA are another alternative methods to produce multiple gRNAs simultaneouslySt-PTG transgenic plants was significantly lower compared to that of St-CP#7 transgenic plants  plants were grown under standard greenhouse conditions. Four virus isolates PVYO-FL (HM367075), PLRV (MT264739.1), PVX (AB056718.1) and PVS (KU896946) were maintained in tobacco or potato plants as inoculated hosts. S. chacoense accession 40-3 containing the Rychc gene that confers extreme resistance to PVY37 was grown under standard greenhouse conditions for comparison.Potato  gene sequence was codon optimized for expression in S. tuberosum (Supplementary Note CP genes (encoding virus coat protein) of four different RNA viruses  . Subsequently, PTG gene with a poly(T) sequence (as a terminator) was cloned into under the Arabidopsis U6 promoter using the restriction enzymes BsaI. Cas13d sequence was cloned as EcoRI/KpnI fragment into the similarly cut vector pJZH131, generating plasmid pEV. Arabidopsis U6 promoter was amplified using pJZH3 as template, and inserted as SacII/SbfI fragment into the pEV vector digested with SacII and SbfI, generating plasmid pXH9. Finally, the PTG sequence was cloned into pXH9 digested with BsaI, producing plasmid pPTG. Sanger sequencing was used to confirm the sequence accuracy of all the clones.Agrobacterium tumefactions strain GV3101 harboring the pPTG construct was employed to transform the potato plants (S. tuberosum cv. Désirée) using a protocol described previously46. Transgenic plants were screened by kanamycin (50 mg L−1) selection and initially tested for the presence of the transgene by PCR assays. Cas13d and gRNA expression levels were subsequently determined by qRT-PCR.2S2O5, 50 mM; Tris-HCl, 125 mM, pH8.8; SDS, 1% (w/v); Glycerol, 10% (v/v)). Proteins were separated on an 8% polyacrylamide gel. Cas13d expression was detected by western blot analysis. An anti-HA antibody  was used as primary antibody (1:3000 dilution). The signals on the membrane were visualized using the enhanced chemiluminescence substrate  following the manufacturer’s instructions.Total proteins were extracted from ~100 mg of leaf samples using protein extraction buffer  according to the manufacturer’s instructions. For virus expression analysis, cDNA was synthesized using reverse transcriptase and oligo(dT) primer . qRT-PCR assays were carried out in a CFX96 Touch TM real-time PCR detection system  using SYBR Premix Ex Taq TM II (TaKaRa). Three technical replicates were performed for each biological replicate. The potato 17.Double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISA) with virus-specific antibodies  were conducted to measure virus accumulation at the protein levels in the systemic leaves according to the manufacturer’s protocolTo determine the response of transgenic plants to various viral strains or isolates, wild-type and transgenic plants were grown under long-day conditions (16-h light: 8-h dark) in the greenhouse at 20–25 °C. Aphid transmission was used for single virus infection except for PVX, which is generally considered transmitted by mechanical inoculation. Aphids were reared on virus-infected wild-type tobacco plants under controlled growth conditions at 20–25 °C. 25 adults were transferred onto wild-type and transgenic potato plants to transmit viruses from the aphids to the plants for 2 days, and then the aphids were removed.47. The mixed infection were also performed by mechanical inoculation using the inocula of the two or three viruses mixed at 1:1 or 1:1:1 titer.For mechanical inoculation, five to eight plants of each line at the 6–8 leaf stage were inoculated with wild-type virus inocula or isolates  by mechanical wounding as described previouslyThe data for virus expression analysis including ELISA and qRT-PCR were analyzed using one-way analysis of variance (ANOVA) coupled with Tukey or Dunnett’s test for multiple comparisons. All the virus assays were repeated at least three independent experiments. Three technical replicates were performed for each biological replicate.Further information on research design is available in the Supplementary MaterialDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary


Canine coronavirus (CCoV) is a positive-strand RNA virus generally responsible for mild-to-severe gastroenteritis in dogs. In recent years, new CCoVs with acquired pathogenic characteristics have emerged, turning the spotlight on the evolutionary potential of CCoVs. To date, two genotypes are known, CCoV type I and CCoV type II, sharing up to 96% nucleotide identity in the genome but highly divergent in the spike gene. In 2009, the detection of a novel CCoV type II, which likely originated from a double recombination event with transmissible gastroenteritis virus (TGEV), led to the proposal of a new classification: CCoV type IIa, including classical CCoVs and CCoV type IIb, including TGEV-like CCoV. Recently, a virus strictly correlated to CCoV was isolated from children with pneumonia in Malaysia. The HuPn-2018 strain, classified as a novel canine–feline-like recombinant virus, is supposed to have jumped from dogs into people. A novel CoV of canine origin, HuCCoV_Z19Haiti, closely related to the Malaysian strain was also detected in a man with fever after travel to Haiti, suggesting that infection with Malaysian-like strains may occur. These data and the emergence of highly pathogenic CoVs in humans underscore the significant threat that CoV spillovers pose to humans and how we should mitigate this hazard. Coronaviridae family with a single-strand positive-sense RNA genome, approximately 27–32kb in size. Based on phylogenetic analyses and genomic structures, CoVs are currently classified within family four genera, Alphacoronavirus, Betacoronavirus, Gammacoronavirus, which is the largest known RNA genome, and Deltacoronavirus, which recognises bats, birds, and likely rodents as natural reservoirs [Alphacoronavirus, which also includes the prototype viruses: feline coronaviruses (FCoVs), transmissible gastroenteritis virus (TGEV), mink coronavirus (MinkCoV), ferret coronavirus (FRCoV), and alpaca respiratory coronavirus [Alphacoronaviruses and Betacoronaviruses, with some exceptions, share an uncleaved protein, but the S protein of Gammacoronaviruses and some Betacoronaviruses is cleaved into S1 and S2 domains by a Golgi-resident protease [Canine coronavirus (CCoV) is an enveloped virus within the servoirs . CCoV beonavirus . The S pprotease . The mosprotease . It contprotease . The N pprotease . The addprotease . Their rprotease .CCoV is considered one of the main pathogens responsible for enteritis in dogs, wolves, foxes, and other canine species , but it Betacoronavirus and Deltacoronavirus; (ii) an additional ORF (referred as ORF3) 624 nt in length and encoding a putative 207aa-protein—likely secreted from the infected cells as no transmembrane region was found—that is completely absent in FCoV type I and of which only remnants remain in the genomes of CCoV type II and TGEV [Starting from its first description in 1971 during an epizootic infection in a canine military unit in Germany , CCoV raand TGEV ,21,22. Eand TGEV . Most deand TGEV .Reports on the emergence of new CCoV strains have always occurred and have been constant over time. Wesley  demonstrThe most striking example of a hypervirulent strain was observed in 2005 in Italy and later in other countries. The detected strain, CB/05, designated pantropic CCoV type II, was associated with the onset of severe and haemorrhagic gastroenteritis, which was sometimes fatal ,28,29,30A genetic analysis of the accessory gene ORF3 highlighted that TGEV originated from CCoV through cross-species transmission, pointing out that the evolution of TGEV, FCoVs, and CCoVs are closely related to each other. In fact, not only did CCoV give rise to FCoV type II through double homologous recombination events between the S and M genes , and recRecently, a study on the characterization of an old CCoV strain (A76 CCoV) was reported . The virRecently, Sha et al.  analysedClinical disease caused by CCoV in dogs is extremely varied, still not fully understood, and with a clinical evolution influenced by many factors, especially by the genetic characteristics of the strains. The virus is responsible for mild-to-moderate enteritis but young pups may develop severe clinical signs, especially when infections with other pathogens occur simultaneously. In addition, coinfections with intestinal viruses that increase the pathogenicity of CCoV, such as canine adenovirus type 1 (CAdV-1) or canine distemper virus (CDV), have been reported ,39. DualIn addition to the classic (mild) enteric CCoVs, several outbreaks of infection characterized by severe clinical signs followed by fatal outcomes are reported. The pantropic CB/05 CCoV type IIa strain isolated in 2005 in Italy caused a systemic disease characterized by high fever, anorexia, lethargy, haemorrhagic diarrhoea, vomiting, leukopenia, and neurological signs with ataxia and seizures. This virulent strain was unexpectedly detected at high titres in the lungs, spleen, liver, kidney, and brain, in addition to the intestinal content . MoreoveCoVs easily cross interspecies barriers and undergo genetic evolution, giving birth to new strains with a selective advantage over the parental genome and characterized by dramatic changes in virulence and tissue tropism . Since cBefore 2002, no in-depth information was available on human CoVs (HCoVs) and their zoonotic potential. The emergence of the first highly pathogenic HCoV in 2002, SARS-CoV-1, the subsequent epidemic Middle East respiratory syndrome CoV, (MERS-CoV), and the pandemic SARS-CoV-2 in 2012 and late 2019, respectively, opened “Pandora’s box” on CoVs, and researchers exploited the extensive knowledge in veterinary medicine on animal CoVs to better understand the characteristics of these viruses and to study their origin . Most reAlphacoronavirus and Betacoronavirus, whilst Gammacoronavirus and Deltacoronavirus were known to infect primarily birds with some mammalian spillover [Deltacoronaviruses [Furthermore, until 2021, reported human infections by CoVs were caused by pillover . Interesaviruses .The occurrence of human epidemics/pandemics caused by SARS-CoV-1, MERS-CoV, and SARS-CoV-2, the detection of CCoV-HuPn-2018 in children with pneumonia in Malaysia, and the first reports of PDCoV infection in Haitian children have raised questions about the zoonotic potential of these viruses and their ability to overcome species barriers.Almost 60% of human pathogens and approximately 60% emerging infectious diseases are zoonoses, and zoonoses transmitted from animals to humans represent an increasingly global public health problem since, despite the fact that animal-to-human transmission has always occurred in the past, its frequency has increased in recent years ,51. CoVs


Ixodes persulcatus tick taken from a human in the south of the Far East was studied. Research was carried out on PEK, Vero, and Vero-E6 cell lines, outbred mice, and chicken embryos using ELISA, PCR, IMFA, plaque formation, and electron microscopy. The tick contained an antigen and a genetic marker of the tick-borne encephalitis virus (TBEV). The patient had post-vaccination antibodies in a titer of 1:200, as a result of which, obviously, an antibody-dependent elimination of TBEV occurred. The tick-borne co-isolate also contained an unknown pathogen (Kiparis-144 virus), which, in our opinion, was a trigger for the activation of chronic infection in suckling white mice. In the laboratory co-isolate, ectromelia virus was present, as evidenced by paw edema during the intradermal infection of mice, characteristic rashes on the chorioallantoic envelope of chicken embryos, and typical plaques on Vero-E6. The Kiparis-144 virus was not pathogenic for white mice and chicken embryos, but it successfully multiplied in the PEK, Vero, and Vero-E6 lines. Viral co-infection was confirmed by electron microscopy. Passaging on mice contributed to an increase in the virulence of the co-isolate, whose titer increased by 10,000 times by the fifth passage, which poses an epidemiological danger.The phenomenon of pathogen co-infection detected in a half-fed  In recent years, in the study of the tick virome, modern research approaches (metagenomic and metatranscriptomic) have been used that have revealed an enormous number of putative new pathogens . Such inPhlebovirus genus of the Bunyaviridae family. This virus has aroused great interest among researchers. It turned out that the SFTS virus is found not only in China but also in the territories adjacent to the Far East—in the Republic of Korea and Japan. In addition, its wide distribution has become known in many countries of the world [Thus, since 2009, severe fever with thrombocytopenia syndrome (SFTS) began to be registered in six provinces of China, the cause of which was not known ,6,7,8,9.he world ,11.Khasan virus (KHAV) was isolated from ticks  collected from spotted deer  in the territory of the Khasansky region (south of the Primorsky Territory of the Far East). Based on biological properties, morphology [Phlebovirus of the Bunyaviridae family [Earlier, in 1971, the rphology ,13, and e family . In thisArteriviridae) in ticks, but they failed to pass LDV. Therefore, the authors of the article characterized only a new strain of ectromelia virus, ECTV (ECTV-WH) [In the last decade, great attention has been drawn to reports on the isolation of especially dangerous pathogens of viral infections. Numerous publications are known that are about new variants of the smallpox virus with non-traditional sources of isolation ,16,17. TECTV-WH) .Ixoodes persulcatus. During the isolation of the putative virus from a half-fed I. persulcatus tick taken from a person in 2016, it was possible to identify the manifestation of co-infection of viral pathogens. It was necessary to verify it and to give a comprehensive biological characterization of viruses.A similar situation arose in our study of a viral isolate from the tick I. persulcatus tick taken on the 5th day of bloodsucking on a 75-year-old patient. The tick bite occurred on 21 May 2016 in the territory of the natural focus in the Nadezhdinsky district (south of the Russian Far East).We isolated an unknown pathogen from the I. persulcatus tick taken from a patient on 21 May 2016 in the south of the Russian Far East (Nadezhda region of Primorsky Krai). In case of primary simultaneous infection in the brain and subcutaneously of 2-day-old outbred white mice of the same litter with a 10% suspension of ticks, on the 6th day, one animal with an unclear clinic of the disease was euthanized, from which the brain was taken for further virological examination. The virus isolates from the 2nd to the 5th passages were used in the work.An unknown-to-us pathogen was isolated from a half-fed The detection of the antigen of the tick-borne encephalitis virus (TBEV) in the tick homogenate was carried out by ELISA using the “VectoTBEV-antigen” kit , according to the instructions of the test system manufacturer.Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and Ehrlichia muris/Ehrlichia chaffeensis, by real-time polymerase chain reaction (RT-PCR) using the kit “AmpliSense TBEV, B. burgdorferi s.l., A. phagocytophilum, E. chaffeensis/E. muris-FL” , according to the manufacturer’s instructions on a cycler with fluorescent detection “ROTOR-GENE Q” .The tick was examined for the presence of genetic markers, such as tick-borne encephalitis virus (TBE), (PEK), Vero, and Vero-E6 culture cells infected with test samples in 3 tubes. Cells from these tubes were collected at certain times of the experiments, and then slides were prepared from the mixture of cells on objective glasses. The antigen of the virus was detected in the cells by applying specific immune serum to the slides and, subsequently, fluorescent immunoglobulins (FITS) in the working dilution specified in the manufacturer’s instructions . Slides were viewed in 3 fields on a fluorescent microscope . The average percentage of cells with the content of the fluorescent virus antigen in relation to the total number of cells in the field of view was considered.The indirect MFA method was used to detect the antigen in Pig embryo kidney PEK, Vero, and Vero-E6 cell lines. The investigation was carried out on 24-well plates using a coating of carboxymethyl cellulose . The titer of virus samples was calculated in plaque-forming units (PFU).The plaque method was used to study the biological characterization of the virus in the 50/mL.The studies were carried out on models of 2-day-old and 4-week-old outbred white mice, which were infected in the brain, subcutaneously and intraperitoneally, with a 10% suspension of the sick mice brain of the 2nd and 5th bioisolate passages, and its titer was determined in lg LDPEK cell line and by detecting the virus titer (PFU) on the Vero-E6 cell line.The study was carried out by infecting 9-day-old chicken embryos with a 0.1 mL virus suspension containing 3 lg PFU on the chorioallantoic envelope (CAE). The results were determined on days 1, 2, and 3 post-infection (pi) by detecting rashes on CAE, as well as by the accumulation of the virus in CAE using the indirect MFA method on the PEK cells, the indirect MFA method was used, and on the Vero-E6 cell line, the plaque method was used. To assess the effect of ultraviolet radiation on the infectious activity of the pathogen, a small Petri dish with a virus-containing liquid (3 lg PFU) that was 3 mm thick was used. This dish was installed at a distance of 24 cm from a source of short-wave ultraviolet rays with a maximum wavelength of 253.7 nm.The influence of physical factors  on the virus inactivation was carried out when exposed to them for 1, 2, 5, and 10 min pi. To verify the results on After filtration through a 0.22 µm millipore filter (Biofil), the study was carried out using the virological methods described above. PEK cell line was infected with these samples, and the supernatant was collected on days 1–3. Virus particles in the supernatants were visualized by negative staining EM analysis. Supernatants from each sample (1 mL) were centrifuged at 3000× g for 20 min at 4 °C to remove cell debris. The clarified supernatants were then centrifuged at 13,000× g for 40 min at 4 °C. The pellets were resuspended in 10 μL PBS. Formvar carbon-coated copper grids were floated in droplets of virus suspension for 10 min and stained with 2% phosphotungstic acid for 1 min at room temperature. Subsequently, the grids were examined by transmission electronic microscopy [Negative contrast electron microscopy was performed on two samples of the isolate. The first sample was the original isolate and the second sample was the isolate after filtration (filter 0.22 nm). The croscopy . The samIxodes persulcatus tick applied to the laboratory for testing on tick-borne infections. The tick was attached in the territory of the Nadezhdinsky region of Primorsky Krai. The tick was removed five days after feeding on the body of the victim. Due to the fact that tick-borne encephalitis virus (TBEV), the first representative of the Flaviviridae family, dominates and is widespread in the Primorsky Territory of the Russian Far East [Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, Ehrlichia muris/Ehrlichia chaffeensis). At the time of the study, in the bioassay of a homogenized tick, an antigen of the TBE virus was detected with a low positivity coefficient (K = 1.7); in real-time qPCR, a genetic marker was determined only for the TBE virus and also with a low indicator (Ct = 34), which indicated a low degree of viral load of this pathogen.In May 2016, a 75-year-old woman with a half-fed Far East , the ticA suspension of the homogenized tick was used to infect the brain of outbred white mice of 2 days of age. The brain of one sick suckling mouse with an unclear clinic was taken on the sixth day pi and passaged also by the intracerebral infection of suckling mice of another family. In the first passage, mice began to get sick for 6 to 10 days, and in the second passage, they were sick for 5–7 days, with the maximum activity of clinical manifestations on the fifth day. In the third passage, all infected suckling mice fell ill on the fourth day, and in the fourth and fifth passages, they fell sick on the third day. It was suggested that the studied isolate could be a TBE virus. However, the study of the sick mice brain of all passages did not show, either in ELISA or in qPCR, the antigen or genetic marker of TBEV. It was necessary to understand the reason for the elimination of TBEV from a bioassay of a half-fed tick, in which, as shown above, low rates of infection with this virus were determined.From the anamnesis, it was found that the patient had previously been vaccinated against TBE. An ELISA study of her blood serum showed the presence of IgG antibodies to the TBE virus in a titer of 1:200. It was suggested that in the process of a tick bloodsucking for 5 days on a vaccinated person, antibodies in such titer could contribute to the neutralization and elimination of the TBE virus in the tick. Thus, in the present case, the tick-borne isolate was obviously not TBEV, but some other unknown virus.Along with this, the study of the blood serum of this woman by the indirect MFA method for the detection of antibodies to this isolate showed the presence of those with a low titer of 1:8 . No clinIn the study of a new unknown pathogen, an important feature is the determination of the sensitivity of various laboratory models to this pathogen. First of all, this refers to the determination of the susceptibility of laboratory animals, in particular white mice, of different age groups with different methods of pathogen inoculation. 50/mL with intracerebral (i/c) infection and 5.6 lg LD50/mL with subcutaneous (s/c) infection. In the model of white mice of 4 weeks of age, weighing 8–10 g, the virus titer upon i/c infection was 6.0 lg LD50/mL, and mice did not become sick with s/c infection (0 lg LD50/mL). After intradermal infection with a viral isolate in the paw of the hind limb, swelling of all paws was observed on days 7–10 due to pronounced diffuse edema, and exudation, skin rashes, and necrotic changes were absent. However, when the viral isolate was passaged up to passage 5, its activity increased sharply, the incubation period was reduced to 3 days, the virus titer increased significantly to 10.0 lg LD50/mL, i.e., 10,000 times. The virulence of the virus isolate also notably increased, and it became pathogenic in all methods of inoculation  pi of E6 cells .PEK culture, in the indirect MFA method, the viral antigen formed a thin fluorescent layer on the cell surface as early as 3 h pi, and a more distinct fluorescence of the cytoplasmic membrane of these cells was observed 24 h later. Less-bright fluorescence was obtained from Vero cells. Vero-E6 cells highly sensitive to the virus isolate showed the brightest fluorescence, especially in their cytoplasm. In the infected culture of these cells, after 3 h, one could observe a bright fluorescence in the cytoplasmic membrane, and after 24 h, it was seen not only the membrane, but also the cytoplasm, indicating the active reproduction of the viral isolate in Vero-E6 cells.This figure shows a bright fluorescence of the antigen in the infected cells of all lines. Despite the fact that the viral isolate did not cause a cytopathic effect and that plaques formed on the PEK cells with the second passage viral isolate, it was detected in the supernatant, as well as in the cell sediment during i/c infection of mice 4 weeks old, its titer was low, and it amounted to 2 lg LD50/0.03 mL and 1.2 lg LD50/0.03 mL, respectively. On this basis, relative to the viral isolate, the PEK line can be classified as a permissive cell culture, but with a low level of productive viral infection.In addition, 5 days pi of PEK cells using the indirect method of fluorescent antibodies (MFA). The results were obtained based on the accumulation of the pathogen in the supernatant of the infected culture, collected in the period from 3 h to 9 days pi, with which the cells of the PEK line were infected. PEK line was infected with samples of supernatants, the fluorescence of cells was detected from the first day pi. The active replication of the isolate and its release into the supernatant were confirmed by positive results in MFA, especially from days 5 to 8, when there was a gradual increase in the number of fluorescent cells  of the infected PEK line.The results obtained above allowed us to continue studying the biological properties of the isolate on Vero-E6 cell line 2, 3, 5, and 7 days pi were previously studied , when exposed to these factors from 1 to 10 min. Observations were made in parallel on two cell lines,  isolate .Vero-E6 cells was observed during infection with samples after exposure to a temperature of 60 °C for 1 and 2 min (7 × 103 PFU and 4 × 101 PFU). With further heating of the viral isolate, the ability to form plaques was lost .The reproduction of the virus in was lost . CompletAs can be seen in A preliminary determination of viral particles size was carried out by filtering the bioisolate through a Millipore filter (0.22 µm). A 10% brain suspension of the sick mice 2 days of age of the fifth passage, taken on the third day pi, was used. At the same time, suckling mice infected in the brain by the resulting filtrate remained healthy for 21 days of observation. Two assumptions were made: mice could not get sick if the virus was larger than 220 nm and it remained on the filter, and they could not get sick if the virus was smaller than 220 nm and it was not pathogenic for outbred suckling mice.Vero-E6 cell line was used  of a chicken embryo.Since the studied isolate was received on the model of outbred white mice, and if the new pathogen had a large size of more than 220 nm, then, first of all, it was necessary to exclude the relationship of the isolate with the mouse ectromelia virus. For this purpose, chicken embryos of 9–10 days of age were infected by introducing a virus-containing material, as well as its filtrate, at a dilution of 10PEK line was infected with a suspension of samples of the chorioallantoic envelope; at three days pi, bright fluorescence was detected in PEK cells in IMPA, indicating the presence of the antigen in samples of both the initial isolate and its filtrate. In addition, the virus titer was determined on the Vero-E6 cell line. The virus isolate at 1 day pi was not detected, and after 3 and 7 days, its titer was 3 lg and 5 lg PFU. In samples of CAE chicken embryos contaminated with filtrate, the virus was not verified during all periods of observation.To prove the specific effect of the virus on chicken embryos, a monolayer of the I. persulcatus contains at least two viruses. To test this hypothesis, it was necessary to receive the direct evidence using the method of electron microscopy.Based on the obtained results of a comprehensive study, it was suggested that the isolate from the tick PEK line infected with the co-isolate, on the third day pi, two types of viral particles were verified, which differed from each other. Two viruses were detected in the sample: one that was large and smallpox-like, up to 500 nm, and the other much smaller, from 60 to 100 nm  was established at low rates in ELISA and qPCR. As it turned out, specific post-vaccination antibodies to TBEV were present in the blood of the examined patient, which probably could contribute to the neutralization and elimination of this pathogen in the tick during bloodsucking. This outcome could be supported by the results of our long-term studies in vitro, ex vivo, and in vivo, aimed at studying the effectiveness of specific protection against the TBE virus [In this work, we also had to solve the issues of the interaction of several pathogens in the microbiome of the tick BE virus . Using aBE virus . This alSuch a classical concept of antibody-dependent elimination of the TBE virus is quite understandable. However, there is an assumption that, in the case of co-infection of the TBE virus and a new virus, the possibility of TBEV elimination can be assumed if the new virus acts as a suppressor during its replication in the tick body. In this regard, it should be noted that for almost two decades we have been observing a gradual decrease in the infection rates of TBEV ticks collected from vegetation in natural foci, as well as ticks attached to people. This downward trend in the TBE virus infection of ticks was reflected in TBE incidence rates in the south of the Far East, where isolated cases of this infection have been recorded in the population in the last decade .Borrelia, and SARS-CoV2 [B. burgdorferi dominates in the human body, causing a long-term chronic course of the disease [Borrelia in the body of a tick infected with the TBEV and B. burgdorferi was previously proven in the experiment [In the case under study, the previously unknown pathogen of the coinfection we identified in the tick microbiome received the opportunity for its replication and the appearance of specific antibodies in the patient, although its titer was low . EarlierARS-CoV2 . Using t disease . Such doperiment .This means that, in cases of the co-infection of different pathogens, any pathogen can dominate. In the described case, when studying the biological characteristics of the viral isolate, we observed an increase in the activity of the mouse pox virus. The isolation of a new virus in ticks can be considered an accident because, as it was found, this virus is not pathogenic for white mice. However, this chance was predetermined by the fact that the isolation of the virus was performed on a model of outbred mice of 2 days of age, which received the ectromelia virus vertically from the mother—the carrier of this chronic infection. We failed to isolate the TBE virus, but an unknown pathogen was identified, which, in our opinion, was a trigger for the activation of a latent chronic infection in sucklings of outbred white mice. A similar case of the coinfection of two viruses (murine ectromelia virus and lactate dehydrogenase-elevating virus) in ticks was described in 2021 [Vero-E6 culture, instability to warming, and resistance to ultraviolet irradiation. However, an electron microscopic examination showed that in addition to the smallpox-like virus, the co-isolate contained another virus that notably differed from the smallpox virus.Using various virological research methods, we focused our efforts on identifying the features of the biological characteristics of the isolated viral co-isolate. In favor of the presence of the ectromelia virus in it, the following manifestations were testified: paw edema during the intradermal infection of adult mice, characteristic rashes on the CAE of infected chicken embryos, typical plaques in the form of an injection on the infected PEK, Vero, and Vero-E6 cell lines. Its presence in the viral co-isolate is shown in the electron microscopy images.Although the new virus we identified turned out to be non-pathogenic for white mice of all age groups and chicken embryos, it did not cause cytopathogenic effects and plaque formation on PEK cells, but it successfully multiplied in “Kiparis-144”. Moreover, the dominant mouse ectromelia virus served as an indicator for the verification of the second virus, Kiparis-144. This is probably why, without such an indicator and without a permissive model for virus isolation, researchers [Based on the obtained results, this viral co-isolate can be characterized as a stable form of the co-cultivation of murine ectromelia virus and a new tick-borne virus, named earchers ,3,20 fai50, i.e., 10,000 times. This indicated that when two viruses were co-cultivated in warm-blooded animals, the new virus acted as an amplifier or “accelerator” of mouse pox virus replication. Their combined interaction was clearly shown by us using electron microscopy (The interaction of these two viruses contributed to a significant increase in the virulence of the viral co-isolate from passages 2 to 5 in outbred white mice with all methods of inoculation. The incubation period in suckling mice infected in the brain was reduced from 5–7 days to 2.5–3 days. The virus titer after the intracerebral infection of 3-week-old mice increased by 4 lg LDcroscopy .Kiparis-144 virus. When bloodsucking ticks with such viruses are found on cows, monkeys, donkeys, and other animals in cases of chronically occurring smallpox virus infection, its activation can occur with the involvement of a human in this process.In epidemiological and epizootological terms, this property of the new virus is a potentially dangerous and naturally causes concern. Moreover, in recent years, natural populations of various representatives of the smallpox virus have become more active in the world ,31,32. WKiparis-144 virus detected in the half-fed tick I. persulcatus in the south of the Far East, as well as to determine the degree of its pathogenicity for humans and animals.Thus, the obtained results allow us to conclude that it is necessary to conduct further scientific research to study the molecular genetic characteristics of not only the ectromelia virus, but also the new


Delia antiqua) can be a devastating pest of onion and related crops around the world. While management of this pest typically relies on insecticides to control these pests, growing resistance and desire for alternative control strategies has motivated a search for new methods to manage them effectively. We investigated the potential of using tiny beneficial roundworms called entomopathogenic nematodes to control onion maggots. We conducted surveys in onion-growing areas to see if these nematodes were naturally present, and also tested their compatibility with commonly used insecticides. Our field trials demonstrated that applying entomopathogenic nematodes can significantly reduce the number of onion plants destroyed by onion maggots, leading to higher crop yields. These nematodes could be a useful tool for onion farmers, whether they practice conventional or organic farming. When combined with other strategies like insecticide seed treatments, the use of entomopathogenic nematodes can provide effective control of onion maggots while boosting crop productivity.Onion maggot (Delia antiqua) is a prominent pest of allium crops in temperate zones worldwide. Management of this pest relies on prophylactic insecticide applications at planting that target the first generation. Because effective options are limited, growers are interested in novel tactics such as deployment of entomopathogenic nematodes. We surveyed muck soils where onions are typically grown to determine if entomopathogenic nematode species were present, and then evaluated the compatibility of entomopathogenic nematode species with the insecticides commonly used to manage D. antiqua. We also evaluated the efficacy of these entomopathogenic nematodes for reducing D. antiqua infestations in the field. No endemic entomopathogenic nematodes were detected in surveys of muck fields in New York. Compatibility assays indicated that, although insecticides such as spinosad and, to some extent, cyromazine did cause mortality of entomopathogenic nematodes, these insecticides did not affect infectivity of the entomopathogenic nematodes. Field trials indicated that applications of entomopathogenic nematodes can reduce the percentage of onion plants killed by D. antiqua from 6% to 30%. Entomopathogenic nematodes reduced D. antiqua damage and increased end of season yield over two field seasons. Applications of entomopathogenic nematodes may be a viable option for reducing D. antiqua populations in conventional and organic systems. Together with other management tactics, like insecticide seed treatments, applications of entomopathogenic nematodes can provide a yield boost and a commercially acceptable level of D. antiqua control.Onion maggot ( Delia antiqua (Meigen) (Diptera: Anthomyiidae) is one of the most important insect pests of onion (Allium cepa L.) worldwide [D. antiqua attacks garlic (Allium sativum L.), scallions (Allium fistulosum L.) and chives (Allium schoenoprasum L.) [D. antiqua can approach 100% without management, most commercial production in temperate regions of the Americas, Europe, and Asia depend on intensive management of this pest [orldwide ,2. In adasum L.) ,4,5. Bechis pest .D. antiqua often involves prophylatic insecticide applications at planting via seed treatments and in-furrow drenches [D. antiqua has three generations per year and the first generation emerges from overwintering pupae in late April and May [D. antiqua may not kill the plant, but can render the bulbs unmarketable.Commercial management of drenches . These t and May ,8,9,10.  and May . FeedingD. antiqua [D. antiqua control. However, chlorpyrifos was banned for use on all crops by the EPA in early 2022. These insecticides tend to only provide control of first-generation D. antiqua, while no insecticides are used to target subsequent generations. Onion growers are interested in non-chemical tactics for managing D. antiqua, especially options that may manage all three generations.Cyromazine and spinosad are two of the most effective insecticides for managing  antiqua ,2. Both  antiqua ,13. UntiD. antiqua generations is entomopathogenic nematodes. Entomopathogenic nematodes are tiny roundworms whose infective juvenile stage seeks out and infects and kills insects [Delia pests. While entomopathogenic nematodes have shown promise in laboratory trials against Delia species such as D. antiqua, D. platura, and D. radicum, field efficacy either was not effective  or n antiqua  and D. p platura ).Delia pests, particularly D. antiqua. After initial introduction, entomopathogenic nematodes can potentially persist and engender long-term control [D. antiqua populations can persist at high levels.Entomopathogenic nematodes have a number of potential advantages for control of  control . This atIn developing entomopathogenic nematodes as biological control agents, species considerations are important. Selecting a virulent entomopathogenic nematode strain that can survive in field conditions is critical. Equally important is making sure that these biological control agents are compatible with current control methods, like soil-applied insecticides. Pesticides used in conjunction with nematodes can have vastly different effects depending on the interaction between the pesticide and the entomopathogenic species used. In some cases, the use of pesticides in conjunction with entomopathogenic nematodes can result in synergistic effects for pest control . In otheD. antiqua in onion, our objectives were to (1) determine if endemic entomopathogenic nematodes are present in muck soils in upstate New York where onions are grown continuously, (2) explore the impact of insecticides commonly used for D. antiqua control on entomopathogenic nematode health and infectivity, (3) to determine if applications of entomopathogenic nematodes in onion fields can reduce onion plant stand loss caused by D. antiqua, and (4) if these entomopathogenic nematodes can persist in these fields for multiple years.To explore the potential integration of biological control into an IPM program for Galleria mellonella larvae, which were used as hosts for entomopathogenic nematodes.To determine if entomopathogenic nematodes occur in muck soils in upstate New York and to examine whether entomopathogenic nematodes were present in our field trial locations, soil cores were collected and then placed in cups containing G. mellonella larvae were added and left under controlled  conditions for seven days. After seven days, G. mellonella larvae were removed, placed on white traps and monitored both for symptomology of entomopathogenic nematode infection and entomopathogenic nematode emergence [G. mellonella larvae were dissected to confirm presence or absence of entomopathogenic nematodes.To evaluate the presence of existing populations of entomopathogenic nematodes in muck soils of New York, 12 onion fields on muck soils were sampled across five counties  in central and western New York. Each of these sites were chosen because of the the presence of muck soils and a history of continuous onion production for multiple years. GPS coordinates can be found in D. antiqua, mortality of entomopathogenic nematode species was assessed after exposure to Entrust SC (a.i. spinosad 22.5%—EPA Reg. No. 62719-621) and Trigard 75WP (a.i. cyromazine 75%—EPA Reg. No. 100-654). To do so, cohorts of entomopathogenic nematode species that had demonstrated efficacy in killing D. antiqua in previous trials were placed in vials containing the insecticide solution and their mortality and ability to infect evaluated after 48hrs.To determine if entomopathogenic nematodes were compatible with insecticides commonly used as onion seed treatments used to control Heterorhabditis bacteriophora, Steinernema diaprepesi, S. feltiae, and S. khuongi. H. bacteriophora and S. feltiae strains were isolated from upstate NY by the Shields lab. S. diaprepesi and S. khuongi strains were isolated from Florida by the LW Duncan lab at University of Florida. All nematode species were subsequently confirmed via barcoding using established techniques [Specifically, a 2 mL solution was prepared containing insecticide treatments and nematode species, with all nematode-pesticide combinations evaluated with 5 replications. Treatments included spinosad, cyromazine, and a water-only control. Entomopathogenic nematode species consisted of chniques . SolutioEntomopathogenic nematode infective juvenile cohorts were left in solution for 48 h in the dark at 25 ± 1 °C. Each cohort (with 5 cohorts each per treatment as described above) was a replication resulting in five replications for each combination of nematode species and treatment. After 48 h, nematodes were triple washed with distilled and deionized water over a fine (325 mesh) screen to remove the pesticide solution and then re-suspended in 2 mL of distilled and deionized water. Three aliquots of 0.1 mL each were placed under a stereoscope for determining mortality of the entomopathogenic nematodes for each replication. Entomopathogenic nematodes were considered dead if they were straight and needle-like under the stereoscope and did not respond to prodding with the tip of a needle.G. mellonella larvae. This was done for each cohort described above resulting in five replications for each combination of nematode species and treatment. These dishes containing entomopathogenic nematode inoculated larvae were left in the dark for 6 days at 25 ± 1 °C. The seventh day after inoculation, any dead larvae were dissected and the presence or absence of entomopathogenic nematodes confirmed under a stereoscope.Immediately following assessments of mortality, 1 mL of the washed and resuspended nematode solution at 1000 IJs/mL was applied to a petri dish (55 mm diameter) containing filter paper and 5 D. antiqua populations and their damage in the field, entomopathogenic nematodes were applied to commercial onion fields on muck soils in Oswego, NY in small plot trials in 2019 and larger scale trials in 2020. Onion plants killed by D. antiqua were evaluated and recorded throughout both seasons using established methodology [To determine if entomopathogenic nematodes could be used to reduce hodology .D. antiqua control trials were sampled within the onion-growing seasons in 2019, 2020, and 2021. Soil cores were taken as above from entomopathogenic nematode-treated and untreated control plots prior to each of three entomopathogenic nematode applications and in the season following applications. This assessment allowed us to determine if: (1) Entomopathogenic nematodes were present in the soil prior to the initiation of our field trial; (2) Entomopathogenic nematodes from previous applications would persist in the soil; and (3) untreated control plots had become contaminated with entomopathogenic nematodes from movement through the soil or transfer on farm implements. Following collection of soil cores, entomopathogenic nematode presence was evaluated as described above using G. mellonella larvae to screen for entomopathogenic nematode infection.The presence of entomopathogenic nematodes in the soil at the location of our A. cepa L.; var. ‘Fortress’ treated with the fungicides penflufen (EverGol Prime) and thiram (Thiram) to protect seedlings against onion smut (Urocystis colchici) and Rhizoctonia spp., respectively. No insecticides were applied at planting. Plots were two rows (approximately 40 cm apart) by 3.5 m long with a 3 m unplanted buffer surrounding plots. Each plot had approximately 100 plants (see data citation for exact numbers). There were two treatments, entomopathogenic nematode treated and untreated, that were replicated four times and arranged in a randomized complete block design. Onions were maintained by the grower following crop production practices that do not impact D. antiqua.This experiment was conducted on muck soil on a commercial farm south of Oswego, New York (GPS: 43°27′04.3″ N 76°23′58.6″ W) in 2019. All experimental plots were planted on 16 May with onion seeds D. antiqua were recorded weekly or every other week in plots until near harvest. The cumulative percentages of onion plants killed by D. antiqua in entomopathogenic nematode-treated plots were compared with those in control plots receiving no application of entomopathogenic nematodes.Onion plants killed by H. bacteriophora and S. feltiae infective juveniles in a 50:50 ratio (each at 1000 IJs/mL) were applied via backpack sprayer over onion rows using 1 L total solution per plot (500 mL/row). The same amount of water (without nematodes) was applied to control plots at the same time. These entomopathogenic nematode species were chosen because of their hardiness to conditions in upstate New York, their complementary stratified preference for soil depths, and their ability to maintain infectivity in the presence of spinosad and cyromazine.Entomopathogenic nematode infective juveniles were applied at a rate of approximately 150 million per acre on 23 May, 6 June, and 1 July. A. cepa L.; var. “Highlander”. Plots were located in muck soils in Oswego County (GPS: 43°27′04.3″ N 76°23′58.6″ W) and were 4 rows (each approximately 40 cm apart) by 30 m with a 5 m buffer between plots on all sides with approximately 1700 plants per plot.In 2020, in a separate location from the 2019 field trials, onion plant stands in entomopathogenic nematode treated plots were compared with control plots receiving no application of entomopathogenic nematodes with four replications in randomized control block design. All experimental plots were planted in late April with onion seeds H. bacteriophora and S. feltiae infective juveniles in a 50:50 ratio (each at 1000 IJs/mL) were applied via backpack sprayer over onion rows using 4 L total solution per plot (1 L/row). The same amount of water (without nematodes) was applied to control plots at the same time. The higher rate in 2020 was selected because of the larger nature of the field trial and because of co-occurrence of seed treatments and conventional D. antiqua management.Entomopathogenic nematode infective juveniles were applied at a rate of approximately 1 billion per acre on 20 April, 16 June, and 6 August . To do so, D. antiqua larvae [D. antiqua infestations. However, FarMore FI500 has not provided a commercially acceptable level of control at this site [During and immediately following the first application, onion stands were evaluated for signs of onion maggot damage (by examining plants for the presence of a larvae ) and stahis site .Data were collated in tabular form  then imported to R V4.2.0 using RStudio as an IDE ,28. The Entomopathogenic nematode presence and absence data from soil surveys are reported as is with no additional analysis applied.2 values. Post-hoc comparisons were made using Dunnett’s test to compare treatment to controls and correct for the family-wise error rate.Linear mixed effects models were used to evaluate the fixed effects of species, pesticide, and their interaction on nematode infective juvenile mortality. Aliquot was included as a random effect to account for repeated measures. Best fit models were chosen based on visual inspection of model diagnostic plots for adherence to model assumptions, analysis of deviance, Akaike and Bayesian information criteria, and pseudo-RG. mellonella larvae. Best fit models were chosen based on visual inspection of model diagnostic plots for adherence to model assumptions, analysis of deviance, Akaike and Bayesian information criteria, likelihood ratio tests, and pseudo-R2 values.Logistic regression was used to evaluate effect of species, pesticide treatment, and their interaction on the ability of exposed cohorts of nematodes to induce mortality in Linear mixed effects models were used to evaluate the fixed effects of entomopathogenic nematode treatment on onion loss due to onion maggot for each field season with date and block included as random effects to account for repeated measures and spatial heterogeneity. Best fit models were selected as above and post-hoc comparisons made with Tukey’s HSD test.D. antiqua in Onion Field TrialsImpact of Entomopathogenic Nematodes on  were conducted, H. bacteriophora and S. feltiae entomopathogenic nematodes were recovered months and a year following application. Following the first three applications in 2019, entomopathogenic nematodes were recovered from sites of application in 2019 and at the beginning of the 2020 field season  . ExposurG. mellonella larvae (S. khuongi ARCA) had an odds ratio significantly different from one  . SpeciesIn both the 2019 and 2020 field season, applications of entomopathogenic nematodes reduced onion plant loss and increased end of season plant stands (in terms of bulb yield) .In small plot field trials in 2019, the effect of entomopathogenic nematode applications was almost significant with the inclusion of all four replicates  [Applications of entomopathogenic nematodes can reduce  killed) . Even inD. antiqua control above and beyond what is currently used by growers.In the 2020 field season in larger plots with conventional insecticide seed treatment and with lower pest populations, applications of entomopathogenic nematodes continued to have a significant effect in reducing pest damage and increasing plant stands. These results suggest that entomopathogenic nematodes could be used to enhance Important to note is that in both field seasons, conventional management continued without alteration; pesticide sprays, fertilizer treatments, herbicide applications were all made by the grower on a regular basis in line with commonly recommended onion production practices in upstate New York. While many factors can cause EPN mortality and reduce efficacy post-application , the abiCompatibility assays with compounds commonly used in seed treatments and potentially lethal to entomopathogenic nematodes also suggest that entomopathogenic nematodes could be a viable and effective control option in field environments. Spinosad caused high mortality in all the entomopathogenic nematode species tested. Cyromazine did not seem to affect mortality to the same extent, similar to results from other work . DespiteD. antiqua is their ability to potentially persist in the field. Surveys of onion muck fields suggested an absence of endemic biological control activity from entomopathogenic nematodes. Inoculating entomopathogenic nematodes into fields from which we were originally unable to detect entomopathogenic nematode activity resulted in establishing a population that we were able to detect a year after first inoculation following harvest, winter, and soil prep for a subsequent season albeit at low numbers. These results suggest that entomopathogenic nematodes could be able to persist in muck soils after initial applications. That being said, recovery of the original population was low in terms of the number of samples from which we were able to detect entomopathogenic nematode populations. The reasons why populations declined over seasons could be due to abiotic factors including weather and frequent disturbance of the soil via tillage and other soil disturbance practices . While populations may persist, they may do so at numbers providing inadequate levels of control. If this were the case, additional applications of entomopathogenic nematodes may be needed to enhance the level of control. Moreover, persistence of entomopathogenic nematodes in non-rotated muck fields where onions are grown continuously for decades may over time substantially reduce D. antiqua populations.Another factor pointing to the viability of entomopathogenic nematodes as an effective management tool for D. antiqua control in both conventional and organic systems. Their ability to improve yield in conjunction with conventional management suggests that they could be an effective control tactic to be included in an integrated pest management approach to control D. antiqua.The ability of entomopathogenic nematodes to survive in muck soils and be compatible with conventional seed treatments suggests that they could be an option for enhancing existing levels of


Idiopathic pulmonary fibrosis (IPF) is a type of interstitial lung disease found mostly in elderly persons, characterized by a high symptom burden and frequent encounters with health services. This study aimed to quantify the economic burden of IPF in Australia with a focus on resource utilization and associated direct costs.Participants were recruited from the Australian IPF Registry (AIPFR) between August 2018 and December 2019. Data on resource utilization and costs were collected via cost diaries and linked administrative data. Clinical data were collected from the AIPFR. A “bottom up” costing methodology was utilized, and the costing was performed from a partial societal perspective focusing primarily on direct medical and non-medical costs. Costs were standardized to 2021 Australian dollars ($).The average annual total direct costs per person with IPF was $31,655 : $27,723–$35,757). Extrapolating costs based on prevalence estimates, the total annual costs in Australia are projected to be $299 million (95% CI: $262 million–$338 million). Costs were mainly driven by antifibrotic medication, hospital admissions and medications for comorbidities. Disease severity, comorbidities and antifibrotic medication all had varying impacts on resource utilization and costs.This cost-of-illness study provides the first comprehensive assessment of IPF-related direct costs in Australia, identifies the key cost drivers and provides a framework for future health economic analyses. Additionally, it provided insight into the major cost drivers which include antifibrotic medication, hospital admissions and medications related to comorbidities. Our findings emphasize the importance of the appropriate management of comorbidities in the care of people with IPF as this was one of the main reasons for hospitalizations.The online version contains supplementary material available at 10.1007/s10198-022-01538-7. Idiopathic pulmonary fibrosis (IPF) is a form of interstitial lung disease (ILD) characterized by progressive fibrosis of the lung parenchyma which results in a progressive decline in lung function, respiratory failure and eventual death . It moreDisease specific therapeutic options for IPF are limited. Only two antifibrotic medications (pirfenidone and nintedanib) have been approved for persons with mild to moderate IPF . AntifibInternationally, there is a limited body of evidence on costs and resource utilization associated with IPF and almost no data from after the approval of antifibrotic treatment . This isThis retrospective cost of illness study was part of a broader study under the National Health and Medical Research Council (NHMRC), Centre for Research Excellence for Pulmonary Fibrosis (CRE-PF) . Costs wParticipants for this study were recruited between August 2018 and December 2019 from the Australian IPF Registry (AIPFR), a national multi-centre, prospective registry of IPF patients facilitated by the Lung Foundation of Australia (LFA) , 11. DetCost diary data were collected via a predesigned paper-based survey instrument, which also collected socio-demographic information, information on treatment and on comorbidities. Clinical information was retrieved from the AIPFR database and included pulmonary function tests (PFT) to ascertain disease severity levels, and body mass index (BMI). Administrative data for health resource utilization and associated costs were obtained via data linkage from the Commonwealth Department of Health, the Centre for Health Record Linkage (CHeReL), the Tasmanian Data Linkage Unit (TDLU) and the Centre for Victorian Data Linkage. Medicare Benefits Schedule (MBS) and Pharmaceutical Benefits Scheme (PBS) were obtained for all participants who consented, while hospital use data were only collected for participants from Victoria (VIC), Tasmania (TAS), New South Wales (NSW), and the Australian Capital Territory (ACT), due to pragmatic considerations regarding multiple ethics applications. Further details on data elements collected can be found in the supplement (Table S1).A number of methods are used to assess disease severity of IPF; however, there is no consensus on a single staging system , 13. PhyThresholds for the FVC% were based on previous studies –17 with In cases where lung function was not available for participants, we categorized participants as “not classified” but still included them in the analysis.For the purpose of this analysis, we classified medications used in the treatment of IPF based on international guidelines . PirfeniCosts are analyzed retrospectively for the 12-month period prior to participants’ recruitment (from August 2018 to December 2019). All costs were standardized to Australian dollars ($) 2021, using the Australian Consumer Price Index from the Australian Bureau of Statistics (ABS) [This study focused primarily on the following four outcome variables: annualized total direct costs, total direct medical costs, total direct non-medical costs and service utilization for major cost drivers. Costs were further stratified by disease severity, participant characteristics and to illustrate the government costs and out of pocket costs (OOP).Prescription medication costs and quantity were extracted from the PBS data and were classified based on the Australian Medicines Handbook . For theNon-prescription medication costs and quantity were extracted from the cost diaries, and these were reported for a period of 4 weeks. Where costs were not recorded for medications, we estimated an average cost, using costs from the four largest pharmacies in Australia . MonthlyAmbulatory care costs and visits were extracted from MBS data. All services that were not flagged as inpatient services were included in this category and included healthcare professionals and medical and diagnostic tests Table . For thiAdmitted patient costs and visits were derived from the Australian Refined Diagnosis Related Groups (AR-DRGs) classification system for public hospital funding published by Independent Hospital Pricing Authority (IHPA . PrivateEmergency department attendances were costed based on Urgency Related Groups (URGs) .Sub-acute admissions  were costed based on the Australian National Subacute and Non-acute Patient Classification (AN-SNAP) .We estimated costs for medical equipment based on participants’ responses in the cost diary. For consumables/accessories where the time frame for use was not provided, an assumed the life span of 1 month was used –26. WherCommunity service attendances and costs were based on participants’ responses in the cost diary. Where costs were not provided by participants, we assumed that services were supplied based on hourly rates, in accordance with the National Summary of Home Care Prices and applicable government subsidies . OOP cosTransportation use and costs were based on responses from the cost diary on distance travelled over a 4-week period that was IPF related. Fuel costs per kilometer were based on average fuel consumption patterns for a passenger vehicle and the average national fuel price for 2019 , 29. Otht test or Chi-squared tests were used where appropriate to compare (i) participants in the study and persons in the AIPFR who did not consent to the study and (ii) participants with PFTs and participants with incomplete or missing PFTs. A p value less than 0.05 was used as a test for statistical significance.Statistical analyses were conducted using R Software . CharactAll costs were summarized as mean costs per person and 95% confidence intervals (95% CI). Summary costs by participants’ characteristics were summarized as mean costs per person (95% CI) and median costs and the interquartile range (IQR). Resource utilization was summarized as the mean (SD) per person and where appropriate as the median per person and IQR.To estimate total annual population costs, we used a prevalence-based approach . PopulatService utilization was summarized as counts and proportions for the 12-month period and means and SDs or 95% CIs where applicable.Generalized linear models (GLM) with gamma distribution and a log link function were used to model the effect of disease severity and other key sociodemographic and clinical variables on total direct costs , 34. Addn = 126) had more comorbidities and were older than participants (Supplement Table S2). Participants with lung function tests (n = 112) were more likely to be on antifibrotic medications (Table Table Eighty-three percent of participants were retired and 80% were aged 65–85 years. The mean age for participants was 73.8 (7.6) years. Most participants were males (61%), Caucasian (90%), resident of major cities (61%) and were based in New South Wales (41%) which is Australia’s most populous state . ApproxiTable A closer evaluation of costs based on participant characteristics Table  demonstrDisaggregation of the total direct costs by medical and non-medical components indicated that direct medical costs accounted for 98% of the total direct costs. Mean direct medical costs and mean direct non-medical costs per person were $31,018  and $637 ($420–$1101), respectively. Seventy-nine percent of direct non-medical costs were OOP costs, with the majority of these attributable to transportation costs  and $32,552  for GAP classification, between $25,124  and $30,784  for FVC classification and between $21,852  and $29,499  for the CPI classification. Table S4 provides further details on selected hypothetical groupings.Table As hospital admissions were of high-cost category, we undertook a more detailed analysis of those. Most admissions were for respiratory diseases (35%), followed by circulatory system diseases (13%) and digestive system diseases (8%). This is in keeping with the comorbidity profile of the participants (Supplement Table S3). Sixty-four percent of all admissions were for comorbidities. The total length of stay (LOS) over the 12-month period for respiratory diseases was 120 days with a mean LOS of 3 days, for circulatory diseases the total number of days was 59 with a mean LOS of 4 days and for digestive system diseases the total was 11 with a mean LOS of 1 day. Nervous system admissions were the fifth most frequent reason for admission but contributed a total of 43 days. On average persons with nervous system disorders were admitted for 7 days, with the longest stay being 28 days. While respiratory disease admissions were the most frequent cause of admission, they did not incur the highest total costs or mean costs per admission. Circulatory system diseases admissions incurred the highest mean costs per admission and total costs. Table A closer look at admissions based on participant characteristics Table  demonstrTable Our study is the first to provide a comprehensive analysis of costs and service utilization for Australians with IPF. Given the importance of nationally relevant economic data for decision making related to health financing and resource allocation in public health, this study has addressed important evidence gaps. Our results demonstrated that the average total direct costs per person for a 12-month period were $31,655, 98% of which were direct medical costs. The total prevalence-based costs of IPF in Australia were $299 million. This cost estimate was mainly driven by antifibrotic medication, medications for comorbidities and hospital admissions. Participants had frequent encounters with GPs and specialists and had numerous diagnostic tests and prescriptions related to comorbidities. Disease severity, comorbidities and antifibrotic medication all had an influence on costs. The main cost driver for direct non-medical costs were transportation costs. Ninety-two percent of costs were incurred by the Australian government with OOP costs accounting for 8% of the total direct costs.We reported total direct costs per person of $31,655 for a 12-month period. When compared to the average per capita health expenditure for Australia for 2019  , our estTotal direct costs were primarily driven by direct medical costs, with antifibrotic medication, hospital admissions and medications for comorbidities being the main cost drivers. There are few published studies which have comprehensively analysed the cost of IPF since the introduction of antifibrotic therapy and those which have, have primarly focused on costs associated with hospitalisations and the associated resource use. Despite this, our study results display general concordance with the limited published literature which identifies transplants, IPF specific medication, admissions and treatment of comorbidities as the key cost generating categories in the management of IPF . Our stuOur results demonstrated that participants had a high number of encounters with GPs and specialists as well as having numerous diagnostic tests and prescriptions related to medications for comorbidities. . PolyphaThe Australian health system was established on the premise of universal access, and this was more than evident in this analysis, as 92% of costs were covered by government sources. Not much literature is available on OOP costs for IPF , but a cOur descriptive statistical analysis demonstrated that disease severity, comorbidities and antifibrotic use had an influence on costs and this was confirmed by the regression analysis. Comorbidities, antifibrotic treatment and increasing disease severity all increased costs. When we looked at resource use and more specifically at hospitalizations which accounted for the highest costs, we saw that comorbidities had the greatest effect. While there was no statistically significant difference between the probability of being admitted whether the participant had a comorbidity or not, when admitted the mean number of admissions was higher in persons with comorbidities than those without. The fact that the odds for admission were not statistically different may be due to improved models of care for persons with chronic complex conditions in Australia including care plans for selfcare and management , use of This study generated cost estimates for a cohort of Australians living with IPF. There were, however, some limitations. First, our sample size was small but given that IPF is a rare and severe disease, this was expected. Based on the estimated number of persons with IPF in Australia, our sample size represents an 8% margin of error with 95% confidence level . This waThis cost-of-illness study is the first study to provide comprehensive direct costs related to IPF management in Australia and provides a framework for future health economics studies/evaluation. It also addresses current evidence gaps which are important for reimbursement and policy decisions related to IPF management. Additionally, it provided insight into the major cost drivers which include antifibrotic medication, hospital admissions and medications related to comorbidities. Furthermore, it emphasizes the importance of early diagnosis, early initiation of antifibrotic therapy and the appropriate management of comorbidities in the care of people with IPF as they all have a significant impact on the well-being of persons living with the disease.Supplementary file1 (DOCX 37 KB)Below is the link to the electronic supplementary material.


Eosinophilic airway inflammation caused by respiratory virus infection has been demonstrated in basic research; however, clinical investigations are lacking. To clarify the extent to which respiratory virus infection induces airway eosinophilic inflammation, we reviewed the results of bronchoalveolar lavage (BAL) and respiratory virus testing performed at our hospital.Among the BAL procedures performed at the University of the Ryukyu Hospital from August 2012 to September 2016, we collected cases of acute respiratory disease in which multiplex polymerase chain reaction (PCR) was used to search for respiratory viruses. The effect of respiratory virus detection on BAL eosinophil fraction was analyzed using statistical analysis. A case study was conducted on respiratory virus detection, which showed an elevated BAL eosinophil fraction.A total of 95 cases were included in this study, of which 17 were PCR-positive. The most common respiratory virus detected was parainfluenza virus (eight cases). The PCR-positive group showed a higher BAL eosinophil fraction than the PCR-negative group (p = 0.030), and more cases had a BAL eosinophil fraction > 3% (p = 0.017). Multivariate analysis revealed that being PCR-positive was significantly associated with BAL eosinophil fraction > 1% and > 3%. There were nine PCR-positive cases with a BAL eosinophil fraction > 1%, of which two cases with parainfluenza virus infection had a marked elevation of BAL eosinophil fraction and were diagnosed with eosinophilic pneumonia.Cases of viral infection of the lower respiratory tract showed an elevated BAL eosinophil fraction. The increase in eosinophil fraction due to respiratory virus infection was generally mild, whereas some cases showed marked elevation and were diagnosed with eosinophilic pneumonia. Respiratory virus infection is not a rare cause of elevated BAL eosinophil fraction and should be listed as a differential disease in the practice of eosinophilic pneumonia. Respiratory viruses are often implicated in the exacerbation of respiratory diseases, including asthma exacerbations, which is a well-known fact . This isIn our institution, when bronchoalveolar lavage (BAL) fluid is collected in clinical practice, multiplex polymerase chain reaction (PCR) testing for respiratory viruses is performed as a routine test to differentiate between infectious. Therefore, PCR results and BAL cell fraction can be collected from past data. We retrospectively summarized this data in patients with acute lung disease and investigate whether the detection of respiratory viruses in BAL fluid is associated with increased BAL eosinophil fraction. Additionally, we reviewed cases of respiratory virus infection and elevated BAL eosinophil fraction.We reviewed data from BAL testing and respiratory virus search multiplex PCR testing of BAL fluid at the University of the Ryukyus Hospital from August 2012 to September 2016. To collect cases of acute respiratory virus infections, cases without respiratory symptoms or new lung lesions within two weeks prior to BAL testing were excluded. Multiplex PCR was performed using commercially available kits , which can detect multiple community-acquired respiratory viruses, such as adenovirus; coronavirus 229E/NL63; OC43; parainfluenza virus 1, 2, 3, 4, rhinovirus A/B/C; respiratory syncytial viruses A and B; metapneumovirus; enterovirus; influenza viruses A and B; and bocavirus 1/2/3/4. We defined elevated BAL eosinophil fraction as a value greater than 1% [Factors that increased BAL eosinophil fraction were statistically analyzed. Statistical analysis was performed using IBM SPSS Statistics for Windows, version 22.0 . Differences among patients’ backgrounds and laboratory test results were compared between the PCR-positive group and the PCR-negative group using the chi-square and Mann–Whitney U tests. Because the PCR-positive group included cases with marked elevation of BAL eosinophil fraction, cutoff values for eosinophil fraction  were set and analyzed with binary variables as well. Stepwise logistic regression analysis (increasing variables method) was performed to explore factors involved in increased BAL eosinophil fraction. The independent variables were PCR-positive in BAL, user of systemic corticosteroid, user of inhaled corticosteroid, bronchial asthma, history of allergies or allergic reactions, and eosinophils in blood > 500/µL. Independent variables were selected based on the results of the univariate analysis and the clinical importance of airway eosinophilic inflammation.This study was approved by the Clinical Research Ethics Committee of the University of the Ryukyus.Ninety-five cases were included in this study, of which 17 were PCR-positive  was conducted. The independent variables were PCR-positive in BAL, user of systemic corticosteroid, user of inhaled corticosteroid, bronchial asthma, history of allergies or allergic reactions, and eosinophils in blood > 500/µL.Of these, nine cases with elevated BAL eosinophil fraction are shown Table . ParainfElevated BAL eosinophil fraction was defined as > 1%. None of the patients were treated with antiviral therapy.Several studies have been conducted on eosinophilic airway inflammation caused by respiratory virus infections. In basic research, respiratory syncytial virus infection is often reported to be involved in eosinophilic airway inflammation –6. CliniEosinophilic airway inflammation caused by respiratory virus infections is considered a protective response to respiratory viruses, and basic medical and clinical studies have shown that eosinophilic inflammation is protective against respiratory viral infections –23. EosiIn the present study, two patients with positive results for parainfluenza virus had a marked elevation of BAL eosinophil fraction and were diagnosed with eosinophilic pneumonia (Case 2 and 3). They had a history of bronchial asthma and had uncontrolled asthma symptoms prior to the examination. Chest imaging revealed a non-regional subpleural infiltrate in the right lower lobe in Case 2, and a typical finding of chronic eosinophilic pneumonia, a photographic negative of pulmonary edema, in Case 3. After bronchoscopy, the patient was diagnosed with eosinophilic pneumonia and started on systemic steroid therapy. Clinical and imaging findings improved quickly, and tapering was completed in about 2 months. Their clinical course was consistent with the usual course of chronic eosinophilic pneumonia. In these two patients, eosinophilic pneumonia was probably caused by parainfluenza infection since there was no other obvious cause. Although there have been reports of cases of eosinophilic pneumonia with a marked elevation of BAL eosinophil fraction in influenza virus infection and COVID-19 –26, therThere were some limitations to this study. First, this was a retrospective single-center study. PCR testing was performed as a routine test on BAL specimens during the study period. However, it was not mandatory and may not have been performed on cases deemed unnecessary by the attending physician. The number of such cases is unknown. It is possible that there was a bias in the backgrounds of the cases included in this study or in the respiratory viruses prevalent in our region. Second, the number of positive cases was too small to properly perform a multivariate analysis of all possible confounders. The statistical analysis on BAL eosinophil fraction should have been performed with the eosinophil fraction measured as a continuous variable; however, we had to replace it with a binary variable to reduce the effect of outliers due to the small population. Third, the degree of eosinophilic airway inflammation is different for different respiratory viruses. A previous report showed that respiratory syncytial virus infection caused higher eosinophil counts than metapneumovirus infection in peripheral blood . When coIn this study, we found that BAL eosinophil fraction was increased in patients infected with respiratory viruses in the lower respiratory tract. The degree of eosinophilic inflammation caused by respiratory virus infections is mild; however, it can occasionally cause eosinophilic pneumonia. Clinicians should also consider respiratory virus infection as a differential cause of elevated BAL eosinophils.