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Topic:Immunology

The equine immune system is a complex network of cells, tissues, and organs that work collaboratively to defend against pathogens and maintain homeostasis. It consists of innate and adaptive components, each with distinct functions and mechanisms. The innate immune system provides the first line of defense through physical barriers, phagocytic cells, and the complement system. The adaptive immune system involves lymphocytes, such as B cells and T cells, which generate specific responses to antigens and provide immunological memory. Research in equine immunology explores the interactions between these components, the impact of genetic and environmental factors on immune function, and the development of vaccines and therapeutics. This page gathers peer-reviewed studies and scholarly articles focusing on the mechanisms, regulation, and clinical applications of the equine immune system in health and disease.
Advanced age in horses affects divisional history of T cells and inflammatory cytokine production.
Mechanisms of ageing and development    September 24, 2008   Volume 129, Issue 11 656-664 doi: 10.1016/j.mad.2008.09.004
Adams AA, Breathnach CC, Katepalli MP, Kohler K, Horohov DW.A number of model systems have been employed to investigate age-associated changes in immune function. The purpose of the current study was to characterize senescent T cells and to investigate the inflamm-aging phenomenon both in vitro and in vivo using the old horse as a model. We examined whether decreased T cell proliferation induced by Con A is caused by increased apoptosis. We also utilized intracellular CFSE to analyze changes within each round of cell proliferation, in particular cytokine production. Intracellular staining with flow cytometry, RT-PCR, and ELISA were used to measure pro-...
The structure of the polysaccharide of the lipopolysaccharide produced by Taylorella equigenitalis type strain (ATCC 35865).
Carbohydrate research    September 24, 2008   Volume 343, Issue 18 3079-3084 doi: 10.1016/j.carres.2008.09.015
Vinogradov E, MacLean LL, Brooks BW, Lutze-Wallace C, Perry MB.Taylorella equigenitalis is a Gram-negative bacterium that causes venereally transmitted contagious equine metritis (CEM), and its identification and differentiation from other bacteria and Taylorella species is an important requirement for the control of CEM infection. Based on the results of NMR and MS analysis, the antigenic O-polysaccharide (O-PS) component of the lipopolysaccharide (LPS) produced by the type strain T. equigenitalis (ATCC 35865) was found to be a linear polymer composed of a repeating disaccharide unit, containing partially amidated 2,3-diacetamido-2,3-dideoxy-alpha-L-gulu...
Equine herpesvirus 1 entry via endocytosis is facilitated by alphaV integrins and an RSD motif in glycoprotein D.
Journal of virology    September 24, 2008   Volume 82, Issue 23 11859-11868 doi: 10.1128/JVI.00868-08
Van de Walle GR, Peters ST, VanderVen BC, O'Callaghan DJ, Osterrieder N.Equine herpesvirus 1 (EHV-1) is a member of the Alphaherpesvirinae, and its broad tissue tropism suggests that EHV-1 may use multiple receptors to initiate virus entry. EHV-1 entry was thought to occur exclusively through fusion at the plasma membrane, but recently entry via the endocytic/phagocytic pathway was reported for Chinese hamster ovary cells (CHO-K1 cells). Here we show that cellular integrins, and more specifically those recognizing RGD motifs such as alphaVbeta5, are important during the early steps of EHV-1 entry via endocytosis in CHO-K1 cells. Moreover, mutational analysis revea...
Activation of peripheral blood monocytes results in more robust production of IL-10 in neonatal foals compared to adult horses.
Veterinary immunology and immunopathology    September 21, 2008   Volume 127, Issue 1-2 167-173 doi: 10.1016/j.vetimm.2008.09.013
Sponseller BA, de Macedo MM, Clark SK, Gallup JM, Jones DE.Foals are particularly vulnerable to infection by Rhodococcus equi during the first 2 weeks of life whereas mature horses are not. While an innate immunodeficiency likely accounts for this clinically relevant vulnerability, the factors that contribute to infection by R. equi have not been fully elucidated. In this study, we demonstrate that cells of the monocyte lineage, including monocytes, macrophages, and dendritic cells, that have been activated with LPS and IFN-gamma, respond with a statistically significant, greater amount of cytokine mRNA production of IL-10, IL-12p35, and IL-12p40 than...
Determination of threshold concentrations of multiple allergenic extracts for equine intradermal testing using normal horses in three seasons.
Veterinary dermatology    September 18, 2008   Volume 19, Issue 5 305-313 doi: 10.1111/j.1365-3164.2008.00696.x
Baxter CG, Vogelnest LJ.Forty-one normal horses were evaluated for reactivity to intradermally injected aqueous allergens to determine allergen threshold concentrations (TC), with potential relevance to equine intradermal testing (IDT). Horses were tested three times over 1 year to assess seasonal variation in reactivity, using three to five serial dilutions of 27 allergens each time. Injection sites were evaluated after 15 min, 1 h, 4 h and 24 h. The highest allergen concentration at which or = 2, scale of 0 to 4) at 15 min was considered the TC. The TC was determined for nine pollens (2000 to > 6000 PNU mL(-1))...
Western Equine Encephalitis submergence: lack of evidence for a decline in virus virulence.
Virology    September 17, 2008   Volume 380, Issue 2 170-172 doi: 10.1016/j.virol.2008.08.012
Forrester NL, Kenney JL, Deardorff E, Wang E, Weaver SC.The incidence of Western Equine Encephalitis (WEE) in humans and equids peaked during the mid-20th century and has declined to fewer than 1-2 human cases annually during the past 20 years. Using the mouse model, changes in WEE virus (WEEV) virulence were investigated as a potential explanation for the decline in the number of cases. Evaluation of 10 WEEV strains representing a variety of isolation locations, hosts, and all decades from the 1940's to the 1990's yielded no evidence of a decline in virulence. These results suggest that ecological factors affecting human and equine exposure should...
A histamine release assay to identify sensitization to Culicoides allergens in horses with skin hypersensitivity.
Veterinary immunology and immunopathology    September 11, 2008   Volume 126, Issue 3-4 302-308 doi: 10.1016/j.vetimm.2008.09.001
Wagner B, Childs BA, Erb HN.Skin hypersensitivity is an allergic disease induced in horses by allergens of Culicoides midges. The condition is typically diagnosed by clinical signs and in some horses in combination with allergy testing such as intradermal skin testing or serological allergen-specific IgE determination. Here, we describe an alternative method for allergy testing: a histamine release assay (HRA) that combines the functional aspects of skin testing with the convenience of submitting a blood sample. The assay is based on the principle that crosslinking of allergen-specific IgE bound via high-affinity IgE rec...
Immunohistochemical localization of oestrogen receptors alpha and beta, progesterone receptor and aromatase in the equine placenta.
Reproduction in domestic animals = Zuchthygiene    September 10, 2008   Volume 44, Issue 2 312-319 doi: 10.1111/j.1439-0531.2008.01073.x
Abd-Elnaeim MM, Derar IR, Wilsher S, Allen WR, Leiser R, Schuler G.The functions of placental oestrogens during equine pregnancy are still unclear. Yet, they may act predominantly as local regulators of growth and differentiation in the microplacentomes. Thus, expression patterns of oestrogen receptors (ERs) alpha and beta were investigated in the microcotyledonary placenta from pregnant mares at 110, 121, 179, 199 and 309 days of gestation by immunohistochemistry. In microplacentomes, both the ER isoforms were detected in trophoblast (T) cells, chorionic villous stroma (FS), microcaruncular epithelium (ME) and microcaruncular stroma (MS). Proportions of posi...
What is your diagnosis? Biopsy impression smear of a hepatic mass in a yearling Thoroughbred filly.
Veterinary clinical pathology    September 3, 2008   Volume 37, Issue 3 339-343 doi: 10.1111/j.1939-165X.2008.00045.x
Gold JR, Warren AL, French TW, Stokol T.A 1-year-old Thoroughbred filly was presented to the Cornell University Hospital for Animals with a 10-day history of fever, diarrhea, inappetance, and hypodipsia. Clinical pathology abnormalities found by the referring veterinarian included erythrocytosis, hyperproteinemia, and increased serum gamma-glutamyltransferase and lactate dehydrogenase activities. At Cornell University, the laboratory abnormalities were confirmed and also included thrombocytosis and hypoglycemia. Erythrocytosis persisted despite vigorous fluid therapy. Ultrasound examination revealed an extremely enlarged liver with ...
An improved method to generate equine dendritic cells from peripheral blood mononuclear cells: divergent maturation programs by IL-4 and LPS.
Immunobiology    September 3, 2008   Volume 213, Issue 9-10 751-758 doi: 10.1016/j.imbio.2008.07.024
Dietze B, Cierpka E, Schäfer M, Schill W, Lutz MB.Equine dendritic cells (eqDC) can be generated from peripheral blood monocytes by propagation in GM-CSF and IL-4. Despite similarities with the generation of human DC, we found significant improvements for eqDC generation and functional influences on eqDC maturation. The fractionation of peripheral blood mononuclear cells (PBMC) by two subsequent gradients at densities of 1.090 and 1.077 as well as an adherence step in AIM V((R)) medium on dishes coated with extracellular matrix components (Primaria) improved the purity and yield of DC. After 3 days, eqDC cultures with GM-CSF alone developed i...
Development of human antibody fragments using antibody phage display for the detection and diagnosis of Venezuelan equine encephalitis virus (VEEV).
BMC biotechnology    September 2, 2008   Volume 8 66 doi: 10.1186/1472-6750-8-66
Kirsch MI, Hülseweh B, Nacke C, Rülker T, Schirrmann T, Marschall HJ, Hust M, Dübel S.Venezuelan equine encephalitis virus (VEEV) belongs to the Alphavirus group. Several species of this family are also pathogenic to humans and are recognized as potential agents of biological warfare and terrorism. The objective of this work was the generation of recombinant antibodies for the detection of VEEV after a potential bioterrorism assault or an natural outbreak of VEEV. Results: In this work, human anti-VEEV single chain Fragments variable (scFv) were isolated for the first time from a human naïve antibody gene library using optimized selection processes. In total eleven different s...
[Seroprevalence and month dynamic of Neospora sp. antibodies in pregnant mares]. Kormann DC, Locatelli-Dittrich R, Richartz RR, Antunes J, Dittrich JR, Patrício LF.In order to evaluate the seroprevalence and the dynamic of anti-Neospora antibodies in pregnant mares, serum samples from 14 animals in the 8th, 9th, 10th and 11th month of pregnancy were analyzed using indirect immunofluorescense technique. Samples diluted 1:50 showed higher seroprevalence on the 8th month (57%) and higher seropositive conversion on the 10th month. 85,7% of the mares were positive for Neospora sp. on at least one month of pregnancy, and seven from that total were positive on the 11th month, three of which showed the highest titles of 1:200 and 1:400. For the samples diluted 1...
[Molecular diagnostic of congenital babesiosis in neonates foals from State of Rio de Janeiro, Brazil]. Santos TM, Santos HA, Massard CL.The aim of this study was to demonstrate, through nested PCR (nPCR) method, the occurrence of congenital babesiosis in two foals born of carrier mares. All mares were positive for T. equi based in visualization of intraerytrocytic parasites in blood smears, in indirect fluorescent antibody test (IFAT) and nPCR reactions. Just one mare was nPCR-positive for B. caballi. After the birth, all foals presented nPCR-positive for T. equi, while just one foal presented nPCR-positive for B. caballi. The present study prove the occurrence of congenital babesiosis in new-born foals, however, new studies a...
[A flow cytometric assay for the expression of interferon gamma in T lymphocytes and its application in the study of EIAV-induced immune response].
Wei sheng wu xue bao = Acta microbiologica Sinica    August 30, 2008   Volume 48, Issue 6 800-805 
Lin Y, Deng X, Shen N, Zhao L, Meng Q, Max J, Wang J, Shao Y, Zhou J.The attenuated vaccine of equine infectious anemia virus (EIAV) is the first lentiviral vaccine that provides solid protection against the infection of EIAV virulent strains. Study of the immune response induced by EIAV vaccine is an important approach to understand the immunity to other lentiviruses. IFN-gamma expressed by specifically stimulated lymphocytes is an important indicator for the evaluation of T cell-mediated immunity. A flow cytometry based assay was established in this study to accurately and effectively detect IFN-gamma expression in different subtypes of T lymphocytes in EIAV-...
Development of an ELA-DRA gene typing method based on pyrosequencing technology.
Tissue antigens    August 26, 2008   Volume 72, Issue 5 464-468 doi: 10.1111/j.1399-0039.2008.01113.x
Díaz S, Echeverría MG, It V, Posik DM, Rogberg-Muñoz A, Pena NL, Peral-García P, Vega-Pla JL, Giovambattista G.The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throug...
Serum antibodies to West Nile virus in naturally exposed and vaccinated horses.
Journal of medical microbiology    August 23, 2008   Volume 57, Issue Pt 9 1087-1093 doi: 10.1099/jmm.0.47849-0
Magnarelli LA, Bushmich SL, Anderson JF, Ledizet M, Koski RA.A polyvalent ELISA and plaque reduction neutralization tests (PRNTs) were used to measure serum antibodies to West Nile virus (WNV) in horses naturally exposed to or vaccinated against this flavivirus in Connecticut and New York State, USA. Relying on a PRNT as a 'gold standard', the main objective was to validate a modified ELISA containing a recombinant WNV envelope protein antigen. It was also important to assess specificity by testing sera from horses that had other, undiagnosed illnesses. Sera for the latter study were obtained from 43 privately owned horses during 1995-1996. Analyses by ...
Equine veterinary journal. Issue content.
Equine veterinary journal    August 16, 2008   Volume 40, Issue 5 430 doi: 10.2746/042516408x327431
Rossdale PD.No abstract available
Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract.
Research in veterinary science    August 15, 2008   Volume 86, Issue 2 189-193 doi: 10.1016/j.rvsc.2008.07.001
Dall'aglio C, Pascucci L, Mercati F, Giontella A, Pedini V, Ceccarelli P.The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques. Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin. Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 rec...
Normal distribution of immunoglobulin isotypes in adult horses.
Veterinary journal (London, England : 1997)    August 9, 2008   Volume 182, Issue 2 359-361 doi: 10.1016/j.tvjl.2008.05.014
de Camargo MM, Kuribayashi JS, Bombardieri CR, Hoge A.Studies focusing on the equine humoral response are scarce, with a bias towards the pre- and post-parturition mare and its foal. The present study attempted to expand current knowledge by establishing normal ranges for adult horse serum isotypes. Immunoglobulin (Ig) concentrations were obtained by screening 47 horses of various breeds and in different training regimes. Radial immunodiffusion values (mg/dL) were 196+/-73 for IgA, 2704+/-1424 for IgG, 419+/-220 for IgG(T) and 70+/-30 for IgM. All values passed the Kolmogorov-Smirnov normality test. The results will be of use to the field veterin...
Kisspeptin immunoreactive neurons in the equine hypothalamus Interactions with GnRH neuronal system.
Journal of chemical neuroanatomy    August 7, 2008   Volume 36, Issue 3-4 131-137 doi: 10.1016/j.jchemneu.2008.07.008
Decourt C, Tillet Y, Caraty A, Franceschini I, Briant C.To determine if kisspeptin could be implicated in the control of reproduction in equine species, we studied the distribution of kisspeptin neurons and their anatomical interactions with GnRH neurons in the hypothalamus of pony mares. Brains were collected in three pony mares between 2 and 4h after ovulation. One major population of kisspeptin immunoreactive cell bodies was found in the arcuate nucleus (ARC), where they extended from the middle of the nucleus to the premammillary recess. Kisspeptin immunoreactive varicose fibers extended from the preoptic area to the mammillary nuclei, with imp...
In vitro and in vivo modulation of the equine immune response by parapoxvirus ovis.
Equine veterinary journal    August 6, 2008   Volume 40, Issue 5 468-472 doi: 10.2746/042516408X322111
Horohov DW, Breathnach CC, Sturgill TL, Rashid C, Stiltner JL, Strong D, Nieman N, Holland RE.While immune modulators are used routinely in equine medicine, their mechanism of action is not always known. Objective: To determine the effect of a commercial preparation of inactivated parapoxvirus ovis (Orf virus; PPVO) on cytokine gene expression by equine peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. Methods: PBMC were prepared from 6 mixed-breed yearlings and cultured in vitro with PPVO with or without Concanavalin A (Con A) for 24 h. Effects on the expression of IFNalpha, IFNbeta IFNgamma, TNFalpha and IL-18 were analysed by real time quantitative PCR (RT-PCR). I...
Evaluation of the pathogenicity of African Horsesickness (AHS) isolates in vaccinated animals.
Vaccine    August 3, 2008   Volume 26, Issue 39 5014-5021 doi: 10.1016/j.vaccine.2008.07.037
von Teichman BF, Smit TK.The polyvalent African Horsesickness (AHS) attenuated live vaccine (ALV) produced by Onderstepoort Biological Products (OBP) Ltd., South Africa, has been associated with some safety concerns and alleged cases of vaccine failure or vaccine-induced disease. The risk of reassortment and reversion to virulence is a common concern associated with the use of ALVs, and a phenomenon reported for viruses with segmented RNA genomes. The purpose of this study was to determine whether or not reassortment of AHS vaccine strains could result in reassortants and reversion to virulence and therefore cause AHS...
Common variable immunodeficiency in horses is characterized by B cell depletion in primary and secondary lymphoid tissues.
Journal of clinical immunology    August 2, 2008   Volume 29, Issue 1 107-116 doi: 10.1007/s10875-008-9221-4
Flaminio MJ, Tallmadge RL, Salles-Gomes CO, Matychak MB.Common variable immunodeficiency (CVID) in horse patients is characterized by late-onset B cell lymphopenia or depletion, hypo- or agammaglobulinemia, impaired humoral response to tetanus toxoid vaccination, and recurrent fevers and bacterial infections. Conclusions: This study describes the clinical and immunologic findings of 14 affected horses (average age 10.7 +/- 4.4 years) of both genders (six females, eight males) and different breeds (eight Thoroughbreds, four Quarter Horses, one Warmblood, one Pony). Serial immunological testing in peripheral blood revealed persistent, severe B cell l...
Denatured virion protein 1 of equine rhinitis B virus 1 contains authentic B-cell epitopes recognised in an enzyme-linked immunosorbent assay–short communication.
Acta veterinaria Hungarica    August 2, 2008   Volume 56, Issue 2 265-270 doi: 10.1556/AVet.56.2008.2.14
Kriegshäuser G, Cullinane A, Kuechler E, Skern T.Equine rhinitis B virus 1 (ERBV1), genus Erbovirus, family Picornaviridae, is a pathogen of horses which causes clinical and subclinical infection of the upper respiratory tract in horses. The virus is widespread in European horse populations and the current standard method for the detection of antibody against ERBV1 is by virus neutralisation (VN). VN tests, however, are labour-intensive and time-consuming, require tissue culture facilities, and generally do not provide same-day results. In this study, a protocol for the high-level expression and purification of recombinant virion protein 1 (...
Report of the 3rd Havemeyer workshop on allergic diseases of the Horse, Hólar, Iceland, June 2007.
Veterinary immunology and immunopathology    July 31, 2008   Volume 126, Issue 3-4 351-361 doi: 10.1016/j.vetimm.2008.07.008
Marti E, Gerber V, Wilson AD, Lavoie JP, Horohov D, Crameri R, Lunn DP, Antczak D, Björnsdóttir S, Björnsdóttir TS, Cunningham F, Dérer M....Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other e...
An equine infectious anemia virus variant superinfects cells through novel receptor interactions.
Journal of virology    July 30, 2008   Volume 82, Issue 19 9425-9432 doi: 10.1128/JVI.01142-08
Brindley MA, Zhang B, Montelaro RC, Maury W.Wild-type strains of equine infectious anemia virus (EIAV) prevent superinfection of previously infected cells. A variant strain of virus that spontaneously arose during passage, EIAV(vMA-1c), can circumvent this mechanism in some cells, such as equine dermis (ED) cells, but not in others, such as equine endothelial cells. EIAV(vMA-1c) superinfection of ED cells results in a buildup of unintegrated viral DNA and rapid killing of the cell monolayer. Here, we examined the mechanism of resistance that is used by EIAV to prevent superinfection and explored the means by which EIAV(vMA-1c) overcomes...
Identification, expression and characterisation of a major salivary allergen (Cul s 1) of the biting midge Culicoides sonorensis relevant for summer eczema in horses.
International journal for parasitology    July 26, 2008   Volume 39, Issue 2 243-250 doi: 10.1016/j.ijpara.2008.06.008
Langner KF, Jarvis DL, Nimtz M, Heselhaus JE, McHolland LE, Leibold W, Drolet BS.Salivary proteins of Culicoides biting midges are thought to play a key role in summer eczema (SE), a seasonal recurrent allergic dermatitis in horses. The present study describes the identification, expression and clinical relevance of a candidate allergen of the North American midge Culicoides sonorensis. Immunoblot analysis of midge saliva revealed a 66 kDa protein (Cul s 1) that was bound by IgE from several SE-affected (SE+) horses. Further characterisation by fragmentation, mass spectrometry and bioinformatics identified Cul s 1 as maltase, an enzyme involved in sugar meal digestion. A c...
Diagnostic equine serology.
The Veterinary clinics of North America. Equine practice    July 26, 2008   Volume 24, Issue 2 311-vi doi: 10.1016/j.cveq.2008.03.002
Zimmerman KL, Crisman MV.This article is presented with two main goals: (1) to provide equine clinicians with a resource for identifying types of serum tests available and (2) to outline briefly the necessary sample type, assay principle, and relative strengths and weakness of the various methods. Specific etiologies are presented and grouped by clinical diagnosis categories, along with brief comments concerning each disorder and its relevant diagnostic assays. This organization provides an abstracted list of infectious disorders commonly considered for the various clinical presentations and a summary of available ser...
Immunodeficiency disorders in horses.
The Veterinary clinics of North America. Equine practice    July 26, 2008   Volume 24, Issue 2 299-vi doi: 10.1016/j.cveq.2008.03.003
Crisman MV, Scarratt WK.Immunodeficiencies are characterized as primary (genetic) or secondary (acquired). Primary immunodeficiencies are relatively uncommon; however, clinically, they present a significant challenge to the practitioner, especially if the underlying disorder goes unrecognized. Secondary immunodeficiencies may present at any age, but failure of passive transfer in neonatal foals is most commonly encountered. This article provides a general overview of clinical signs and diagnosis of primary and secondary immunodeficiencies currently recognized in horses.
Binding of equine infectious anemia virus to the equine lentivirus receptor-1 is mediated by complex discontinuous sequences in the viral envelope gp90 protein.
The Journal of general virology    July 18, 2008   Volume 89, Issue Pt 8 2011-2019 doi: 10.1099/vir.0.83646-0
Sun C, Zhang B, Jin J, Montelaro RC.The identification and characterization of a functional cellular receptor for equine infectious anemia virus (EIAV), designated equine lentivirus receptor-1 (ELR1), a member of the tumour necrosis factor receptor protein family, has been reported previously [Zhang, B. et al. (2005). Proc Natl Acad Sci U S A, 102 , 9918-9923]. The finding of a single receptor for EIAV is distinct from feline, simian and human immunodeficiency viruses, which typically utilize two co-receptors for infection, but is similar to avian and murine oncoviruses, which use single receptors. This study sought to determine...
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