Mixed infection of FeLV-A and FeLV-B caused more severe anemia and death in infected cats

Mixed infection of FeLV-A and FeLV-B caused more severe anemia and death in infected cats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mixed infection of FeLV-A and FeLV-B caused more severe anemia and death in infected cats Xinyue Zhang, Chenchen Ma, Haibing Dong, Wenjuan Wu, Jianhao Shi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7850769/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Feline leukemia virus (FeLV) exhibits remarkable subtype diversity and is associated with fatal outcomes including anemia and lymphoma. However, there is a virtual void of FeLV-related research in China. In this study, 536 samples were detected using the qPCR method, followed by the amplification of the env gene sequences for genetic evolution and recombination analyses. Our results demonstrated a 2.2% positivity rate (12/536), with successful env gene amplification from four FeLV-A and FeLV-B positive samples. Phylogenetic reconstruction revealed that Chinese FeLV-A strains likely originated from multiple countries. Importantly, recombination analysis confirmed FeLV-B emergence through genetic recombination between FeLV-A and enFeLV. Electron microscopy visualized characteristic FeLV particles, while necropsy and histopathological examination identified lymphoma-like lesions. In conclusion, these findings reveal that FeLV-A strains pose a risk of transmission in China. They can recombine with enFeLV to generate FeLV-B with enhanced pathogenicity, leading to severe anemia in infected cats and accelerating their death process. FeLV qPCR phylogenetic analysis recombination analysis anemia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Feline leukaemia virus (FeLV) is an enveloped single-stranded RNA virus, which belongs to the family Retroviridae, genus Gammaretrovirus (Hartmann 2012 ; Watanabe et al. 2013 ). The genome is approximately 8.4 kb in length, consisting of three main genes (gag, pol, and env) flanked by long terminal repeats (LTRs) composed of U3, R, and U5 regions (Hartmann and Hofmann-Lehmann 2020 ). The FeLV virions are spherical particles approximately 100 nm in diameter and represent the primary causative agent of feline leukemia (FeL) (Willett and Hosie 2013 ). The characteristic manifestations of feline leukemia include malignant lymphoma, immunosuppression, and bone marrow failure-induced anemia (Mackey et al. 1975 ). FeLV infection patterns are categorized into four types: abortive, progressive, regressive, and focal infections. Persistent viremia represents the hallmark of progressive infection, which typically carries a poor prognosis (Hartmann and Hofmann-Lehmann 2020 ). Horizontal transmission primarily occurs through mutual grooming, shared food/water sources, and aggressive interactions among cats, with viral shedding predominantly via oronasal secretions, urine, and feces (Hardy et al. 1975 ; Gomes-Keller et al. 2009 ). FeLV can be classified into two major categories: non-pathogenic endogenous feline leukemia virus (enFeLV) and pathogenic exogenous feline leukemia virus (exFeLV). enFeLV can integrate into the host genome and is vertically transmitted through Mendelian inheritance (Polani et al. 2010 ). Although enFeLV itself cannot produce infectious viral particles, it readily recombines with exFeLV to generate pathogenic variants (Coffin 2004 ; Powers et al. 2018 ). Currently, it can be divided into six subgroups: FeLV-A, B, C, D, E and T. FeLV-A is the most prevalent subtype, capable of horizontal transmission with relatively low pathogenicity (Chiu et al. 2018 ). FeLV-B is frequently associated with lymphoma cases in cats, with current evidence suggesting its pathogenicity requires FeLV-A as a helper virus (Sheets et al. 1993 ; Ahmad and Levy 2010 ). FeLV-C, the rarest subtype, demonstrates a strong correlation with aplastic anemia (Shelton and Linenberger 1995 ). FeLV continues to circulate as a major pathogenic threat to domestic and wild felids across all continents. However, FeLV-related research in China remains virtually unexplored. The present study aimed to perform the molecular characterization of FeLV-positive samples and determine the circulating viral subtype in cats from China, as well as to evaluate phylogenetic relations. 2. Materials and Methods 2.1 Sample collection and nucleic acid extraction During 2023–2024, our laboratory collected a total of 536 samples across 12 provinces in China. The samples were obtained from animal hospitals, catteries, and stray animal rescue centers. Detailed information including gender, age, body weight, breed, and clinical symptoms was recorded for each specimen. The sample types included EDTA-anticoagulated whole blood, oronasal swabs, and pathological tissue materials. Genomic RNA was isolated from 536 samples using the Viral DNA/RNA Extraction Mini Kit (Mabio, GuangZhou, China) according to the manufacturer’s instructions. 2.2 qPCR detection of exFeLV For detection of exFeLV, the amplification of a region within the U3 LTR sequences was performed (Table 1 ). The 20-µL reaction consisted of 0.3 µL of each primer and probe, 10 µL of 2 × One Step U + Mix, 1 µL One Step U + Enzyme Mix (Vazyme, China), 3.1 µL of PCR grade H 2 O, and 5 µL of sample RNA. The qPCR cycling conditions were as follows: 48°C for 10 min, 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 15 s (Torres et al. 2005 ). Table 1 Primers for amplification of FeLV-A, and FeLV-B env segments FeLV type Primer sequence Primer location Product References exFeLV Forward AGTTCGACCTTCCGCCTCAT U3 LTR 68 bp (Torres et al. 2005 ) Reverse AGAAAGCGCGCGTACAGAAG U3 LTR Probe TAAACTAACCAATCCCCATGCCTCTCGC U3 LTR FeLV-A Forward ACCCAAGCTAATGCCACCTC FeLV-A env 1.9 kb (Erbeck et al. 2021 ) Reverse CCTCTAACTTCCTTGTATCTCATGG FeLV-A 3′LTR FeLV-B Forward CAGATCAGGAACCATTCCCAGG enFeLV env, FeLV-B 1.8 kb Reverse CCTCTATCTTCCTTGTATCTCATGG FeLV-A, FeLV-B 3′LTR 2.3 Amplification of exFeLV env gene For amplification of FeLV-A, B env fragments, using different specific primers (Table 1 ). PCR was performed using 4 µL genomic RNA in 50 µL reactions, with One step Enzyme Mix (Vazyme, China). The PCR conditions were 50°C for 30 min, 94°C for 3 min, 35 cycles of denaturing at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 2 min, and a final extension at 72°C for 5 min (Erbeck et al. 2021 ). The positive samples were sent to Beijing Tsingke B iotechnology Co., Ltd. for sequencing. 2.4 Phylogenetic analysis and subtypes genetic identification A total of 10 FeLV subtype reference sequences were downloaded from the NCBI database (Table 2 ). The env nucleotide sequences was aligned using the MegAlign module in DNASTAR Lasergene 7. The phylogenetic trees were constructed using Molecular Evolutionary Genetics (MEGA; version 11.0) software with the maximum likelihood (ML) method, and the accession number of the viruses are labelled in the phylogenetic trees. Table 2 Sequence information of reference virus strains GenBank accession no. Reference strain Region Year Subtype OR227280 1180/2021_A Italy 2021 FeLV-A MF681666 FeLV_US_x2004R1_Pco2012 American 2012 FeLV-A EU629217 328A-MG Brazil 2008 FeLV-A KP728112 Glasgow-1 United Kingdom 2015 FeLV-A LC765238 FeLV-A_ON-T_1–1 Japan 2023 FeLV-A EU629220 918B-MG Brazil 2008 FeLV-B J03448 lambda-B1 United States 1993 FeLV-B K01208 ST United States 1993 FeLV-B AY364319 FeLV-GGAG United States 2003 enFeLV LC196055 enFeLV-clone3 Japan 2016 enFeLV 2.5 Recombination analysis In this study, we utilized RDP software (version 4.0) to analyze potential recombination events within the env gene, employing a comprehensive set of seven computational methods including RDP, GENECONV, BootScan, MaxChi, Chimera, SiScan, and 3 eq to identify potential recombinant events. Identified as recombinant strains by meeting four or more of the above methods ( p < 0.05). 2.6 Analysis of positive sample FJ-1 2.6.1 Clinical examination Collect basic clinical information of FJ-1, perform blood routine, biochemical index test, and imaging examination. 2.6.2 Necropsy and Histopathology observations Post-euthanasia necropsy of FJ-1 was performed with owner consent. Fresh samples of the spleen, liver, intestines, and lymph nodes were promptly immersed in 10% neutral buffered formalin for fixation. Following fixation, tissues were trimmed into appropriately sized blocks for embedding and subsequently stained with hematoxylin and eosin (H&E). 2.6.3 Microscopy observations For the positive samples, spleen tissues were collected, mixed and then grinded in phosphatebuffered saline (PBS) to prepare 10% (wt/vol) suspension. After being frozen and thawed three times, the suspension was centrifuged at 12,000×g for 10 min and subsequently filtered through 0.22 µm filters (Millipore, Bedford, MA, USA). 2.6.4 Whole Genome Sequencing and Recombination Analysis of FJ-1 The spleen tissue of FJ-1 was homogenized, subjected to repeated freeze-thaw cycles, and centrifuged to collect the supernatant. Nucleic acids were then extracted and stored on dry ice before being transported to Shandong Senqi Biotechnology Co., Ltd. for whole-genome sequencing. Recombination events were validated using SimPlot software (version 3.5.1). A sliding window of 200 nucleotides with a step size of 20 nucleotides was applied to analyze recombination between the FJ-1B strain and its two parental strains (FJ-1A and enFeLV). 3. Results 3.1 qPCR detection of exFeLV We collected 536 samples and tested them for exFeLV using TaqMan qPCR, and exFeLV antigen-positive reactions were detected 2.2% (12/536) of the samples. Their basic clinical characteristics are presented in Table 3 . Table 3 Information of FeLV qPCR-positive samples Sample number Year Wight(kg) Sex Life history Result / CT value Clinical symptoms FJ-1 7Y 4.3 Male Outdoor +/9.13 Anemia, Suspected lymphoma CQ-1 2Y5M 4.56 Male Outdoor +/12.89 Anemia, Stomatitis SH-5 2Y 3.76 Male Outdoor +/17.42 Anemia HZ-3 1Y 2.1 Male Indoor +/12.1 Anemia WY-1 5Y6M 5.5 Male Outdoor +/33.26 - CQ-3 2Y9M 4.73 Male Outdoor +/33.96 Stomatitis FS-1 1Y 5.8 Female Outdoor +/29.53 - LY-2 8M 2.86 Male Outdoor +/34.7 Stomatitis WX-1 8Y 3.6 Fale Outdoor +/29.50 - QD-2 9Y 3.1 Female Indoor +/30.36 - QD-36 2Y 5 Male Outdoor +/31.27 - QD-47 1Y5M 4.47 Male Indoor +/29.75 - Note: *, FJ-1, CQ-1, SH-5, and HZ-3 all showed symptoms of anemia. They died within six months of diagnosis after ineffective treatment. The symbol “-” indicates the absence of obvious clinical symptoms. 3.2 Amplification of exFeLV env gene Env gene amplification was performed on 12 FeLV qPCR-positive samples, identifying 4 cases of FeLV-A and FeLV-B coinfection (Fig. 1 ). The sequence was confirmed as exFeLV by NCBI database alignment analysis. 3.3 Sequence alignment and phylogenetic analysis Phylogenetic analysis indicated that the partial nucleotide sequences of the env gene of strain CQ-1A were closely related to the Japanese strain (LC765238), while strains FJ-1A, HZ-3A, and SH-5A were more closely related to the American strain (MF681666) and the Italian strain (OR227280). These four FeLV-A positive strains belong to the same monophyletic group as the reference strains of subtype A, and thus are classified into the FeLV-A subgroup. The partial nucleotide sequences of the env gene of strain CQ-1B have a relatively close genetic relationship with the reference strains of subtype B. Together with other strains (FJ-1B, HZ-3B, and SH-5B), they formed multiple clades, showing a relatively high divergence rate. These four FeLV-B positive strains belong to the same monophyletic group as the reference strains of subtype B, and are thus classified into the FeLV-B subgroup. Figure 2 presents the phylogenetic tree and corresponding subgroups. 3.4 Recombination analysis Recombination analysis of strain HZ-3B and its parental strains HZ-3A and enFeLV (LC196055) was performed using RDP software. The results showed that the first 800 bp nucleotide sequence of the env gene of the HZ-3B strain was similar to that of enFeLV, while the last 1000 bp nucleotide sequence was similar to that of HZ-3A (Fig. 3 ). The recombination events were highly reliable (p < 0.05), demonstrating statistical significance. 3.5 Analysis of positive samples FJ-1 3.5.1 Clinical examination The FJ-1 sample was collected from a cat suffering from severe anemia and suspected lymphoma. Both clinical and laboratory tests were positive for exFeLV, with mixed FeLV-A and FeLV-B infection, and a Ct value of 9.13. Upon visual examination, FJ-1 appeared emaciated with abdominal distension. X-ray and ultrasound examinations revealed a rounded liver edge and severe splenomegaly (Fig. 4 ). Blood routine examination shows a decrease in the number of platelets and red blood cells, accompanied by a decline in hemoglobin and hematocrit, suggesting the presence of anemia (Table 4 ). Biochemical index detection shows that the index of blood urea nitrogen (BUN: 13 mg/dL) is decreased, while the indices of alkaline phosphatase (ALKP: 212 U/L), gamma-glutamyl transferase (GGT: 5 U/L) and total bilirubin (TBIL: 4.2 mg/dL) are increased, indicating the presence of anemia and liver abnormalities Table 4 FJ-1 Blood Indicator report card Parameter Result Unit Range RBC L 2.24 10 12 /L 4.60–10.20 HGB L 43 g/L 85–153 HCT L 12.3 % 26.0–47 MCV H 55.0 fL 38.0–54.0 MCH H 19.2 pg 11.8–18.0 MCHC 349 g/L 290–380 RDW-CV H 31.1 % 16.0–23.0 RDW-SD H 67.5 fL 26.4–43.1 PLT L 62 10 9 /L 100–518 MPV 11.1 fL 9.9–16.3 PDW 14.7 % 12.0-17.5 PCT L 0.068 % 0.090–0.700 3.5.2 Necropsy and Histopathology observations Sample FJ-1 was obtained from a male cat presenting with severe anemia and suspected lymphoma. Due to poor response to treatment and following the owner’s request, the cat was euthanized. Gross pathology revealed marked splenomegaly (30 cm in length, 0.39 kg in weight), diffuse hepatic lipidosis (0.4 kg in weight), and mesenteric lymphadenopathy with abnormal hyperplasia (Fig. 5 ). Histopathological examination of the lymph nodes and small intestine sections showed lymphocytes with convoluted nuclei and chromatin projections, forming intranuclear elongated divisions, suggestive of neoplastic transformation (Fig. 6 ). 3.5.3 Microscopy observations Transmission electron microscopy of FJ-1 revealed numerous spherical, enveloped virions measuring approximately 100 nm in diameter displaying the characteristic morphology of FeLV (Fig. 7 ). 3.5.4 Whole Genome Sequencing and Recombination Analysis of FJ-1 The FJ-1A and FJ-1B sequences of about 8500 bp were amplified by whole gene sequencing, respectively. Similarity analysis of the query strain (FJ-1B) and comparison strains (FJ-1A and enFeLV) was performed using SimPlot software. The recombination region of FJ-1B is involved in three genes, Gag, Pol and Env (Fig. 8 ) 4. Discussion This study conducted phylogenetic and recombination analyses of FeLV-A and B strains prevalent in China. FeLV-A formed relatively concentrated branches on the genetic phylogenetic tree, while FeLV-B formed multiple branches, showing a high divergence rate. The results of the recombination analysis further supported the conclusion that FeLV-B was generated by the recombination of FeLV-A and enFeLV. Among the cats that tested positive by qPCR, 83.3% (10/12) were male and 75.0% (9/12) had an outdoor stray history. This may be attributed to male cats' higher likelihood of outdoor fights over territory and mating (Gleich et al. 2009 ; Biezus et al. 2019 , 2023 ). Notably, exFeLV (FeLV-A, B) gene sequences were successfully amplified by RT-PCR in only 4 of the 12 qPCR-positive samples, with no detectable amplification in the remaining 8 samples. This persisted despite our attempts using primers from multiple literature sources, increasing viral load, optimizing annealing temperatures, and employing higher-efficiency enzymes. Clinical situation of the 4 positive samples showed that FeLV-A infection alone was clinically insignificant, in latency or present in low concentrations within the host, clinically insignificant and not easily detected (high Ct values by qPCR), but created an opportunity to recombine with enFeLV in the body, which recombined to produce FeLV-B, and the mixture of the two infections caused more severe anemia and death of the infected cats. In addition, all 4 cats coinfected with FeLV-A and B exhibited common hematological abnormalities: the number of red blood cells was less than 2.2×10¹²/L (reference value: 4.6–10.2×10¹²/L), the hemoglobin concentration was lower than 43 g/L (reference value: 85–153 g/L), the hematocrit was below 12.3% (reference value: 26–47%), and the number of platelets was less than 62×10⁹/L (reference value: 100–518×10⁹/L), while WBC counts remained within normal range. These findings align precisely with reported hematologic changes in progressive FeLV infection (Gleich and Hartmann 2009 ). Previous studies indicate that cats in the progressive infection stage have shorter survival times and poor treatment outcomes, with FeLV-B infection often diagnosed in young cats showing severe clinical symptoms. In this study, FeLV-B infection was detected in all 4 RT-PCR positive samples. These cats were in the progressive infection stage and died successively within half a year following ineffective treatment, consistent with previous findings. In addition, FeLV-B was only detected in the presence of FeLV-A, further supporting the rarity of horizontal transmission of FeLV-B alone (Erbeck et al. 2021 ; Coelho et al. 2008 ; Stewart et al. 2013 ). Declarations Conflict of interest : The authors declare that there are no conflicts of interest. The funders had no role in the design of this study, analyses, interpretation of the data, or decision to publish. Funding: This study was supported by the Suzhou AiYi Animal Pharmaceutical Co. (No20220605) and the Key R&D Program Project of Shandong Province: ResearchDevelopment and Applicationine-source Functional Pet Food (No.2024TSGC0166). Author Contribution Y.Y., Q.M., H.Y. and C.Z. designed the experiments. X.Z., C.M. and H.D. performed the experiments. W.W., J.S. and Y.L. analyzed the data. X.Z. and C.M. wrote the ar-ticle. 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Vet J 195:16–23. https://doi.org/10.1016/j.tvjl.2012.07.004 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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15:12:17","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":302211,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/d217ce8f595ac26611139220.png"},{"id":95035839,"identity":"123fad8c-011c-4d05-a8f9-125a47a3a294","added_by":"auto","created_at":"2025-11-03 15:12:17","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58710,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/7be299b4c73d7a4b9187a7db.png"},{"id":95035846,"identity":"b8aa66cf-e388-4ea6-bbf1-09f8c37c0c9b","added_by":"auto","created_at":"2025-11-03 15:12:17","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83508,"visible":true,"origin":"","legend":"","description":"","filename":"4c9e60681c3b4dd5bcb43aabacd72a891structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/c26f2cd45ab8840b4e1ce000.xml"},{"id":95221827,"identity":"422b542f-51e5-4225-a85a-3e741016222c","added_by":"auto","created_at":"2025-11-05 16:19:45","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":89497,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/d5b8a34b0334a4a616d41143.html"},{"id":95222517,"identity":"df74d6e1-2fb7-4d7e-ae83-dd1ac8c040e4","added_by":"auto","created_at":"2025-11-05 16:20:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":213131,"visible":true,"origin":"","legend":"\u003cp\u003eRT-PCR products of the FJ-1 env gene. M, DNA Marker DL-5000; N, negative control; 1, FJ-1A (FeLV-A); 3, FJ-1B (FeLV-B).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/338e29919cd9eabf3b8e7490.png"},{"id":95035820,"identity":"f23eb602-cd4f-4bfb-9a05-fae2352cb499","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":243376,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis based on the nucleotide sequence of the env gene. The evolutionary history was inferred using the Neighbor-Joining method. Solid red circles indicate FeLV strains from cats with severe anemia in this study.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/781ec493542d88c2cdcf52ed.png"},{"id":95222648,"identity":"1334807c-ff59-489a-9f5c-952002f9fe41","added_by":"auto","created_at":"2025-11-05 16:20:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1332967,"visible":true,"origin":"","legend":"\u003cp\u003eRecombination analysis results of the HZ-3 env gene. Recombinant strain: HZ-3B; Parental strain: HZ-3A, enFeLV (LC196055).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/c78b28565de2b07d788ccc7f.png"},{"id":95035822,"identity":"e56b445c-68d7-420d-9178-036d9ab16f90","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":304055,"visible":true,"origin":"","legend":"\u003cp\u003eResults of imaging and pathologic changes. (\u003cstrong\u003eA\u003c/strong\u003e) X-ray: abdominal expansion of the spleen (dotted line); (\u003cstrong\u003eB\u003c/strong\u003e) ultrasound: blunt rounding of the liver margins (red arrowhead).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/8d27b751b67dc7901a4567ac.png"},{"id":95035823,"identity":"1f9ef4b8-3a37-4476-91ec-59dcc8f659d2","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":385442,"visible":true,"origin":"","legend":"\u003cp\u003eNecropsy findings of FJ-1. (\u003cstrong\u003eA\u003c/strong\u003e) Congested and enlarged spleen up to 30 cm long, weighing 0.39 kg; (\u003cstrong\u003eB\u003c/strong\u003e) Diffuse lipid deposits in the liver, weighing 0.4 kg; (\u003cstrong\u003eC\u003c/strong\u003e) Enlarged lymph nodes (red dashed delimitation) and some abnormal parenchymal hyperplasia (black dashed delimitation).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/ccf3f40046ade9ed4138a3c0.png"},{"id":95035825,"identity":"51e0a203-ea3a-401b-bdfd-c1f0fef3a90c","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1139638,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological characterization of lymph nodes and small intestine in FJ-1. (\u003cstrong\u003eA\u003c/strong\u003e) Lymph nodes; (\u003cstrong\u003eB\u003c/strong\u003e) small intestine. lymphocytes with convoluted nuclei and chromatin projections forming long divisions in the nucleus (arrowhead). (HE, 400x).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/5e1dda72fd5a2344033df223.png"},{"id":95035829,"identity":"9d3466d6-4b43-4101-a8c9-804c99c1421c","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1972839,"visible":true,"origin":"","legend":"\u003cp\u003eElectron microscopy observation results of the FJ-1.\u003cstrong\u003e \u003c/strong\u003eVirus particles are spherical and about 100 nm in diameter (arrowhead).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/7525134d5eaabb260ef32575.png"},{"id":95035827,"identity":"c1faee33-6b55-4909-963f-c60d5d94778c","added_by":"auto","created_at":"2025-11-03 15:12:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1425194,"visible":true,"origin":"","legend":"\u003cp\u003eRecombination analysis of the FeLV-B (FJ-1) variant. FJ-1B had four main recombination regions from the enFeLV strain. Similarity analysis was performed with the query strain (FJ-1B) and the comparison strains (FJ-1A and enFeLV). The analysis was constructed with the following parameters: window: 200 bp, step: 20 bp, gap strip: On, Kimura (2-parameter), T/t: 2.0. The four recombination regions are shown with dotted lines.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/32189382405ef6cb0887bf54.png"},{"id":95524138,"identity":"fa206238-7957-41e1-b2b4-856aefe2623f","added_by":"auto","created_at":"2025-11-10 10:02:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8795932,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7850769/v1/30d87798-5a3c-457d-86fb-1e94adf2b60d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mixed infection of FeLV-A and FeLV-B caused more severe anemia and death in infected cats","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFeline leukaemia virus (FeLV) is an enveloped single-stranded RNA virus, which belongs to the family Retroviridae, genus Gammaretrovirus (Hartmann \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Watanabe et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The genome is approximately 8.4 kb in length, consisting of three main genes (gag, pol, and env) flanked by long terminal repeats (LTRs) composed of U3, R, and U5 regions (Hartmann and Hofmann-Lehmann \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The FeLV virions are spherical particles approximately 100 nm in diameter and represent the primary causative agent of feline leukemia (FeL) (Willett and Hosie \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The characteristic manifestations of feline leukemia include malignant lymphoma, immunosuppression, and bone marrow failure-induced anemia (Mackey et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1975\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFeLV infection patterns are categorized into four types: abortive, progressive, regressive, and focal infections. Persistent viremia represents the hallmark of progressive infection, which typically carries a poor prognosis (Hartmann and Hofmann-Lehmann \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Horizontal transmission primarily occurs through mutual grooming, shared food/water sources, and aggressive interactions among cats, with viral shedding predominantly via oronasal secretions, urine, and feces (Hardy et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1975\u003c/span\u003e; Gomes-Keller et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFeLV can be classified into two major categories: non-pathogenic endogenous feline leukemia virus (enFeLV) and pathogenic exogenous feline leukemia virus (exFeLV). enFeLV can integrate into the host genome and is vertically transmitted through Mendelian inheritance (Polani et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Although enFeLV itself cannot produce infectious viral particles, it readily recombines with exFeLV to generate pathogenic variants (Coffin \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Powers et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Currently, it can be divided into six subgroups: FeLV-A, B, C, D, E and T. FeLV-A is the most prevalent subtype, capable of horizontal transmission with relatively low pathogenicity (Chiu et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). FeLV-B is frequently associated with lymphoma cases in cats, with current evidence suggesting its pathogenicity requires FeLV-A as a helper virus (Sheets et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Ahmad and Levy \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). FeLV-C, the rarest subtype, demonstrates a strong correlation with aplastic anemia (Shelton and Linenberger \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1995\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFeLV continues to circulate as a major pathogenic threat to domestic and wild felids across all continents. However, FeLV-related research in China remains virtually unexplored. The present study aimed to perform the molecular characterization of FeLV-positive samples and determine the circulating viral subtype in cats from China, as well as to evaluate phylogenetic relations.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Sample collection and nucleic acid extraction\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eDuring 2023\u0026ndash;2024, our laboratory collected a total of 536 samples across 12 provinces in China. The samples were obtained from animal hospitals, catteries, and stray animal rescue centers. Detailed information including gender, age, body weight, breed, and clinical symptoms was recorded for each specimen. The sample types included EDTA-anticoagulated whole blood, oronasal swabs, and pathological tissue materials.\u003c/p\u003e\u003cp\u003eGenomic RNA was isolated from 536 samples using the Viral DNA/RNA Extraction Mini Kit (Mabio, GuangZhou, China) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 qPCR detection of exFeLV\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor detection of exFeLV, the amplification of a region within the U3 LTR sequences was performed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The 20-\u0026micro;L reaction consisted of 0.3 \u0026micro;L of each primer and probe, 10 \u0026micro;L of 2 \u0026times; One Step U\u0026thinsp;+\u0026thinsp;Mix, 1 \u0026micro;L One Step U\u0026thinsp;+\u0026thinsp;Enzyme Mix (Vazyme, China), 3.1 \u0026micro;L of PCR grade H\u003csub\u003e2\u003c/sub\u003eO, and 5 \u0026micro;L of sample RNA. The qPCR cycling conditions were as follows: 48\u0026deg;C for 10 min, 95\u0026deg;C for 3 min, followed by 40 cycles of 95\u0026deg;C for 5 s and 60\u0026deg;C for 15 s (Torres et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers for amplification of FeLV-A, and FeLV-B env segments\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFeLV type\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrimer sequence\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrimer location\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eProduct\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReferences\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eexFeLV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGTTCGACCTTCCGCCTCAT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eU3 LTR\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e68 bp\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e(Torres et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2005\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAGAAAGCGCGCGTACAGAAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eU3 LTR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eProbe\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTAAACTAACCAATCCCCATGCCTCTCGC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eU3 LTR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eACCCAAGCTAATGCCACCTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFeLV-A env\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.9 kb\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003e(Erbeck et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCTCTAACTTCCTTGTATCTCATGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFeLV-A 3\u0026prime;LTR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eFeLV-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCAGATCAGGAACCATTCCCAGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eenFeLV env, FeLV-B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e1.8 kb\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eCCTCTATCTTCCTTGTATCTCATGG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFeLV-A, FeLV-B 3\u0026prime;LTR\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Amplification of exFeLV env gene\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor amplification of FeLV-A, B env fragments, using different specific primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). PCR was performed using 4 \u0026micro;L genomic RNA in 50 \u0026micro;L reactions, with One step Enzyme Mix (Vazyme, China). The PCR conditions were 50\u0026deg;C for 30 min, 94\u0026deg;C for 3 min, 35 cycles of denaturing at 94\u0026deg;C for 30 s, annealing at 58\u0026deg;C for 30 s, and extension at 72\u0026deg;C for 2 min, and a final extension at 72\u0026deg;C for 5 min (Erbeck et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The positive samples were sent to Beijing Tsingke B iotechnology Co., Ltd. for sequencing.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Phylogenetic analysis and subtypes genetic identification\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eA total of 10 FeLV subtype reference sequences were downloaded from the NCBI database (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The env nucleotide sequences was aligned using the MegAlign module in DNASTAR Lasergene 7. The phylogenetic trees were constructed using Molecular Evolutionary Genetics (MEGA; version 11.0) software with the maximum likelihood (ML) method, and the accession number of the viruses are labelled in the phylogenetic trees.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSequence information of reference virus strains\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGenBank accession no.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eReference strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eRegion\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSubtype\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eOR227280\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1180/2021_A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eItaly\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2021\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMF681666\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeLV_US_x2004R1_Pco2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eAmerican\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2012\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEU629217\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e328A-MG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBrazil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eKP728112\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGlasgow-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnited Kingdom\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2015\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLC765238\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeLV-A_ON-T_1\u0026ndash;1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJapan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-A\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eEU629220\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e918B-MG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eBrazil\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-B\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eJ03448\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003elambda-B1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnited States\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-B\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eK01208\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eST\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnited States\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1993\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eFeLV-B\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAY364319\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFeLV-GGAG\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eUnited States\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2003\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eenFeLV\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLC196055\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eenFeLV-clone3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eJapan\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2016\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eenFeLV\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Recombination analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eIn this study, we utilized RDP software (version 4.0) to analyze potential recombination events within the env gene, employing a comprehensive set of seven computational methods including RDP, GENECONV, BootScan, MaxChi, Chimera, SiScan, and 3 eq to identify potential recombinant events. Identified as recombinant strains by meeting four or more of the above methods (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Analysis of positive sample FJ-1\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Clinical examination\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eCollect basic clinical information of FJ-1, perform blood routine, biochemical index test, and imaging examination.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Necropsy and Histopathology observations\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePost-euthanasia necropsy of FJ-1 was performed with owner consent. Fresh samples of the spleen, liver, intestines, and lymph nodes were promptly immersed in 10% neutral buffered formalin for fixation. Following fixation, tissues were trimmed into appropriately sized blocks for embedding and subsequently stained with hematoxylin and eosin (H\u0026amp;E).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 Microscopy observations\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eFor the positive samples, spleen tissues were collected, mixed and then grinded in phosphatebuffered saline (PBS) to prepare 10% (wt/vol) suspension. After being frozen and thawed three times, the suspension was centrifuged at 12,000\u0026times;g for 10 min and subsequently filtered through 0.22 \u0026micro;m filters (Millipore, Bedford, MA, USA).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.6.4 Whole Genome Sequencing and Recombination Analysis of FJ-1\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe spleen tissue of FJ-1 was homogenized, subjected to repeated freeze-thaw cycles, and centrifuged to collect the supernatant. Nucleic acids were then extracted and stored on dry ice before being transported to Shandong Senqi Biotechnology Co., Ltd. for whole-genome sequencing.\u003c/p\u003e\u003cp\u003eRecombination events were validated using SimPlot software (version 3.5.1). A sliding window of 200 nucleotides with a step size of 20 nucleotides was applied to analyze recombination between the FJ-1B strain and its two parental strains (FJ-1A and enFeLV).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 qPCR detection of exFeLV\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eWe collected 536 samples and tested them for exFeLV using TaqMan qPCR, and exFeLV antigen-positive reactions were detected 2.2% (12/536) of the samples. Their basic clinical characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eInformation of FeLV qPCR-positive samples\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSample number\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eYear\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWight(kg)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eSex\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLife history\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eResult / CT value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eClinical symptoms\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFJ-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/9.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAnemia,\u003c/p\u003e\u003cp\u003eSuspected lymphoma\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCQ-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2Y5M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/12.89\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAnemia, Stomatitis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSH-5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/17.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAnemia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHZ-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIndoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/12.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAnemia\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWY-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5Y6M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/33.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCQ-3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2Y9M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/33.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eStomatitis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFS-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/29.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLY-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/34.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eStomatitis\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eWX-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e8Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/29.50\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQD-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e9Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIndoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/30.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQD-36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2Y\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOutdoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/31.27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eQD-47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e1Y5M\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4.47\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eIndoor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e+/29.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: *, FJ-1, CQ-1, SH-5, and HZ-3 all showed symptoms of anemia. They died within six months of diagnosis after ineffective treatment. The symbol \u0026ldquo;-\u0026rdquo; indicates the absence of obvious clinical symptoms.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Amplification of exFeLV env gene\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eEnv gene amplification was performed on 12 FeLV qPCR-positive samples, identifying 4 cases of FeLV-A and FeLV-B coinfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sequence was confirmed as exFeLV by NCBI database alignment analysis.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Sequence alignment and phylogenetic analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003ePhylogenetic analysis indicated that the partial nucleotide sequences of the env gene of strain CQ-1A were closely related to the Japanese strain (LC765238), while strains FJ-1A, HZ-3A, and SH-5A were more closely related to the American strain (MF681666) and the Italian strain (OR227280). These four FeLV-A positive strains belong to the same monophyletic group as the reference strains of subtype A, and thus are classified into the FeLV-A subgroup.\u003c/p\u003e\u003cp\u003eThe partial nucleotide sequences of the env gene of strain CQ-1B have a relatively close genetic relationship with the reference strains of subtype B. Together with other strains (FJ-1B, HZ-3B, and SH-5B), they formed multiple clades, showing a relatively high divergence rate. These four FeLV-B positive strains belong to the same monophyletic group as the reference strains of subtype B, and are thus classified into the FeLV-B subgroup. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents the phylogenetic tree and corresponding subgroups.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Recombination analysis\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRecombination analysis of strain HZ-3B and its parental strains HZ-3A and enFeLV (LC196055) was performed using RDP software. The results showed that the first 800 bp nucleotide sequence of the env gene of the HZ-3B strain was similar to that of enFeLV, while the last 1000 bp nucleotide sequence was similar to that of HZ-3A (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The recombination events were highly reliable (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), demonstrating statistical significance.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Analysis of positive samples FJ-1\u003c/h2\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e3.5.1 Clinical examination\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe FJ-1 sample was collected from a cat suffering from severe anemia and suspected lymphoma. Both clinical and laboratory tests were positive for exFeLV, with mixed FeLV-A and FeLV-B infection, and a Ct value of 9.13. Upon visual examination, FJ-1 appeared emaciated with abdominal distension. X-ray and ultrasound examinations revealed a rounded liver edge and severe splenomegaly (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eBlood routine examination shows a decrease in the number of platelets and red blood cells, accompanied by a decline in hemoglobin and hematocrit, suggesting the presence of anemia (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Biochemical index detection shows that the index of blood urea nitrogen (BUN: 13 mg/dL) is decreased, while the indices of alkaline phosphatase (ALKP: 212 U/L), gamma-glutamyl transferase (GGT: 5 U/L) and total bilirubin (TBIL: 4.2 mg/dL) are increased, indicating the presence of anemia and liver abnormalities\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eFJ-1 Blood Indicator report card\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eResult\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUnit\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eRange\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRBC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e12\u003c/sup\u003e/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.60\u0026ndash;10.20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHGB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e43\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eg/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e85\u0026ndash;153\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.0\u0026ndash;47\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003efL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e38.0\u0026ndash;54.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e19.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003epg\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.8\u0026ndash;18.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMCHC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e349\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eg/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e290\u0026ndash;380\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRDW-CV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e31.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e16.0\u0026ndash;23.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRDW-SD\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eH\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e67.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003efL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e26.4\u0026ndash;43.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePLT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e62\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e10\u003csup\u003e9\u003c/sup\u003e/L\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e100\u0026ndash;518\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMPV\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003efL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e9.9\u0026ndash;16.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePDW\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e12.0-17.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePCT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eL\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.068\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e%\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.090\u0026ndash;0.700\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.5.2 Necropsy and Histopathology observations\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSample FJ-1 was obtained from a male cat presenting with severe anemia and suspected lymphoma. Due to poor response to treatment and following the owner\u0026rsquo;s request, the cat was euthanized. Gross pathology revealed marked splenomegaly (30 cm in length, 0.39 kg in weight), diffuse hepatic lipidosis (0.4 kg in weight), and mesenteric lymphadenopathy with abnormal hyperplasia (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eHistopathological examination of the lymph nodes and small intestine sections showed lymphocytes with convoluted nuclei and chromatin projections, forming intranuclear elongated divisions, suggestive of neoplastic transformation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.5.3 Microscopy observations\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eTransmission electron microscopy of FJ-1 revealed numerous spherical, enveloped virions measuring approximately 100 nm in diameter displaying the characteristic morphology of FeLV (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section3\"\u003e\u003ch2\u003e3.5.4 Whole Genome Sequencing and Recombination Analysis of FJ-1\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe FJ-1A and FJ-1B sequences of about 8500 bp were amplified by whole gene sequencing, respectively. Similarity analysis of the query strain (FJ-1B) and comparison strains (FJ-1A and enFeLV) was performed using SimPlot software. The recombination region of FJ-1B is involved in three genes, Gag, Pol and Env (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThis study conducted phylogenetic and recombination analyses of FeLV-A and B strains prevalent in China. FeLV-A formed relatively concentrated branches on the genetic phylogenetic tree, while FeLV-B formed multiple branches, showing a high divergence rate. The results of the recombination analysis further supported the conclusion that FeLV-B was generated by the recombination of FeLV-A and enFeLV.\u003c/p\u003e\u003cp\u003eAmong the cats that tested positive by qPCR, 83.3% (10/12) were male and 75.0% (9/12) had an outdoor stray history. This may be attributed to male cats' higher likelihood of outdoor fights over territory and mating (Gleich et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Biezus et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Notably, exFeLV (FeLV-A, B) gene sequences were successfully amplified by RT-PCR in only 4 of the 12 qPCR-positive samples, with no detectable amplification in the remaining 8 samples. This persisted despite our attempts using primers from multiple literature sources, increasing viral load, optimizing annealing temperatures, and employing higher-efficiency enzymes.\u003c/p\u003e\u003cp\u003eClinical situation of the 4 positive samples showed that FeLV-A infection alone was clinically insignificant, in latency or present in low concentrations within the host, clinically insignificant and not easily detected (high Ct values by qPCR), but created an opportunity to recombine with enFeLV in the body, which recombined to produce FeLV-B, and the mixture of the two infections caused more severe anemia and death of the infected cats.\u003c/p\u003e\u003cp\u003eIn addition, all 4 cats coinfected with FeLV-A and B exhibited common hematological abnormalities: the number of red blood cells was less than 2.2\u0026times;10\u0026sup1;\u0026sup2;/L (reference value: 4.6\u0026ndash;10.2\u0026times;10\u0026sup1;\u0026sup2;/L), the hemoglobin concentration was lower than 43 g/L (reference value: 85\u0026ndash;153 g/L), the hematocrit was below 12.3% (reference value: 26\u0026ndash;47%), and the number of platelets was less than 62\u0026times;10⁹/L (reference value: 100\u0026ndash;518\u0026times;10⁹/L), while WBC counts remained within normal range. These findings align precisely with reported hematologic changes in progressive FeLV infection (Gleich and Hartmann \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePrevious studies indicate that cats in the progressive infection stage have shorter survival times and poor treatment outcomes, with FeLV-B infection often diagnosed in young cats showing severe clinical symptoms. In this study, FeLV-B infection was detected in all 4 RT-PCR positive samples. These cats were in the progressive infection stage and died successively within half a year following ineffective treatment, consistent with previous findings. In addition, FeLV-B was only detected in the presence of FeLV-A, further supporting the rarity of horizontal transmission of FeLV-B alone (Erbeck et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Coelho et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Stewart et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflict of interest :\u003c/h2\u003e\u003cp\u003eThe authors declare that there are no conflicts of interest. The funders had no role in the design of this study, analyses, interpretation of the data, or decision to publish.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis study was supported by the Suzhou AiYi Animal Pharmaceutical Co. (No20220605) and the Key R\u0026amp;D Program Project of Shandong Province: ResearchDevelopment and Applicationine-source Functional Pet Food (No.2024TSGC0166).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.Y., Q.M., H.Y. and C.Z. designed the experiments. X.Z., C.M. and H.D. performed the experiments. W.W., J.S. and Y.L. analyzed the data. X.Z. and C.M. wrote the ar-ticle. H.Y.and C.Z. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAhmad S, Levy LS (2010) The frequency of occurrence and nature of recombinant feline leukemia viruses in the induction of multicentric lymphoma by infection of the domestic cat with FeLV-945. Virology 403:103\u0026ndash;110. https://doi.org/10.1016/j.virol.2010.04.011\u003c/li\u003e\n \u003cli\u003eBiezus G, Grima de Cristo T, Bassi das Neves G, et al (2023) Phylogenetic identification of feline leukemia virus A and B in cats with progressive infection developing into lymphoma and leukemia. Virus Res 329:199093. https://doi.org/10.1016/j.virusres.2023.199093\u003c/li\u003e\n \u003cli\u003eBiezus G, Machado G, Ferian PE, et al (2019) Prevalence of and factors associated with feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) in cats of the state of Santa Catarina, Brazil. Comp Immunol Microbiol Infect Dis 63:17\u0026ndash;21. https://doi.org/10.1016/j.cimid.2018.12.004\u003c/li\u003e\n \u003cli\u003eChiu ES, Hoover EA, VandeWoude S (2018) A Retrospective Examination of Feline Leukemia Subgroup Characterization: Viral Interference Assays to Deep Sequencing. Viruses 10:29. https://doi.org/10.3390/v10010029\u003c/li\u003e\n \u003cli\u003eCoelho FM, Bomfim MRQ, de Andrade Caxito F, et al (2008) Naturally occurring feline leukemia virus subgroup A and B infections in urban domestic cats. J Gen Virol 89:2799\u0026ndash;2805. https://doi.org/10.1099/vir.0.2008/003855-0\u003c/li\u003e\n \u003cli\u003eCoffin JM (2004) Evolution of retroviruses: fossils in our DNA. Proc Am Philos Soc 148:264\u0026ndash;280\u003c/li\u003e\n \u003cli\u003eErbeck K, Gagne RB, Kraberger S, et al (2021) Feline Leukemia Virus (FeLV) Endogenous and Exogenous Recombination Events Result in Multiple FeLV-B Subtypes during Natural Infection. J Virol 95:e0035321. https://doi.org/10.1128/JVI.00353-21\u003c/li\u003e\n \u003cli\u003eGleich S, Hartmann K (2009) Hematology and serum biochemistry of feline immunodeficiency virus-infected and feline leukemia virus-infected cats. J Vet Intern Med 23:552\u0026ndash;558. https://doi.org/10.1111/j.1939-1676.2009.0303.x\u003c/li\u003e\n \u003cli\u003eGleich SE, Krieger S, Hartmann K (2009) Prevalence of feline immunodeficiency virus and feline leukaemia virus among client-owned cats and risk factors for infection in Germany. J Feline Med Surg 11:985\u0026ndash;992. https://doi.org/10.1016/j.jfms.2009.05.019\u003c/li\u003e\n \u003cli\u003eGomes-Keller MA, G\u0026ouml;nczi E, Grenacher B, et al (2009) Fecal shedding of infectious feline leukemia virus and its nucleic acids: a transmission potential. Vet Microbiol 134:208\u0026ndash;217. https://doi.org/10.1016/j.vetmic.2008.08.011\u003c/li\u003e\n \u003cli\u003eHardy WD, Hess PW, Essex M, et al (1975) Horizontal transmission of feline leukemia virus in cats. Bibl Haematol 67\u0026ndash;74. https://doi.org/10.1159/000397519\u003c/li\u003e\n \u003cli\u003eHartmann K (2012) Clinical aspects of feline retroviruses: a review. Viruses 4:2684\u0026ndash;2710. https://doi.org/10.3390/v4112684\u003c/li\u003e\n \u003cli\u003eHartmann K, Hofmann-Lehmann R (2020) What\u0026rsquo;s New in Feline Leukemia Virus Infection. Vet Clin North Am Small Anim Pract 50:1013\u0026ndash;1036. https://doi.org/10.1016/j.cvsm.2020.05.006\u003c/li\u003e\n \u003cli\u003eMackey L, Jarrett W, Jarrett O, Laird H (1975) Anemia associated with feline leukemia virus infection in cats. J Natl Cancer Inst 54:209\u0026ndash;217. https://doi.org/10.1093/jnci/54.1.209\u003c/li\u003e\n \u003cli\u003ePolani S, Roca AL, Rosensteel BB, et al (2010) Evolutionary dynamics of endogenous feline leukemia virus proliferation among species of the domestic cat lineage. Virology 405:397\u0026ndash;407. https://doi.org/10.1016/j.virol.2010.06.010\u003c/li\u003e\n \u003cli\u003ePowers JA, Chiu ES, Kraberger SJ, et al (2018) Feline Leukemia Virus (FeLV) Disease Outcomes in a Domestic Cat Breeding Colony: Relationship to Endogenous FeLV and Other Chronic Viral Infections. J Virol 92:e00649-18. https://doi.org/10.1128/JVI.00649-18\u003c/li\u003e\n \u003cli\u003eSheets RL, Pandey R, Jen WC, Roy-Burman P (1993) Recombinant feline leukemia virus genes detected in naturally occurring feline lymphosarcomas. J Virol 67:3118\u0026ndash;3125. https://doi.org/10.1128/JVI.67.6.3118-3125.1993\u003c/li\u003e\n \u003cli\u003eShelton GH, Linenberger ML (1995) Hematologic abnormalities associated with retroviral infections in the cat. Semin Vet Med Surg Small Anim 10:220\u0026ndash;233\u003c/li\u003e\n \u003cli\u003eStewart H, Jarrett O, Hosie MJ, Willett BJ (2013) Complete genome sequences of two feline leukemia virus subgroup B isolates with novel recombination sites. Genome Announc 1:e00036-12. https://doi.org/10.1128/genomeA.00036-12\u003c/li\u003e\n \u003cli\u003eTorres AN, Mathiason CK, Hoover EA (2005) Re-examination of feline leukemia virus: host relationships using real-time PCR. Virology 332:272\u0026ndash;283. https://doi.org/10.1016/j.virol.2004.10.050\u003c/li\u003e\n \u003cli\u003eWatanabe S, Kawamura M, Odahara Y, et al (2013) Phylogenetic and structural diversity in the feline leukemia virus env gene. PLoS One 8:e61009. https://doi.org/10.1371/journal.pone.0061009\u003c/li\u003e\n \u003cli\u003eWillett BJ, Hosie MJ (2013) Feline leukaemia virus: half a century since its discovery. Vet J 195:16\u0026ndash;23. https://doi.org/10.1016/j.tvjl.2012.07.004\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"FeLV, qPCR, phylogenetic analysis, recombination analysis, anemia","lastPublishedDoi":"10.21203/rs.3.rs-7850769/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7850769/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFeline leukemia virus (FeLV) exhibits remarkable subtype diversity and is associated with fatal outcomes including anemia and lymphoma. However, there is a virtual void of FeLV-related research in China. In this study, 536 samples were detected using the qPCR method, followed by the amplification of the env gene sequences for genetic evolution and recombination analyses. Our results demonstrated a 2.2% positivity rate (12/536), with successful env gene amplification from four FeLV-A and FeLV-B positive samples. Phylogenetic reconstruction revealed that Chinese FeLV-A strains likely originated from multiple countries. Importantly, recombination analysis confirmed FeLV-B emergence through genetic recombination between FeLV-A and enFeLV. Electron microscopy visualized characteristic FeLV particles, while necropsy and histopathological examination identified lymphoma-like lesions. In conclusion, these findings reveal that FeLV-A strains pose a risk of transmission in China. They can recombine with enFeLV to generate FeLV-B with enhanced pathogenicity, leading to severe anemia in infected cats and accelerating their death process.\u003c/p\u003e","manuscriptTitle":"Mixed infection of FeLV-A and FeLV-B caused more severe anemia and death in infected cats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 15:12:12","doi":"10.21203/rs.3.rs-7850769/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ffffa14d-7a17-4850-827e-3e30aea10d9e","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-06T20:53:14+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-03 15:12:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7850769","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7850769","identity":"rs-7850769","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}