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Short Communication |
1 Department of Medicine and Epidemiology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
2 Department of Medicine, Infectious Diseases Division, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA
Correspondence
Ellen E. Sparger
eesparger{at}ucdavis.edu
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vif). Virus in peripheral blood, antiviral antibody or CD4 T-cell count alterations were not detected in kittens inoculated with FIV-pPPRvif plasmid, with the exception of one kitten that demonstrated FIV Gag antibody production at 42 weeks after inoculation. In contrast, wild-type FIV-pPPR-infected kittens were viraemic, seropositive and exhibited a decrease in the CD4 T-cell subset in peripheral blood. Interestingly, FIV-specific T-cell proliferative responses detected at 32 and 36 weeks after infection were comparable for both FIV-pPPRvif- and wild-type FIV-pPPR-inoculated kittens and suggested the possibility of a discreet tissue reservoir supporting sustained FIV-pPPRvif expression or replication. Overall, these findings confirmed that the severe virus attenuation for both replication and pathogenicity exhibited by a vif-deleted FIV mutant is similar for both neonatal and adult hosts.
Present address: Department of Surgery, Duke University Medical Center, Duke University, Durham, NC 27710, USA. 
Present address: IDEXX Laboratories, 2825 KOVR Drive, West Sacramento, CA 95605, USA.
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; Gabuzda et al., 1992; Gupta et al., 2007; Harmache et al., 1996; Inoshima et al., 1996; Kristbjornsdottir et al., 2004; Lockridge et al., 1999; Paul et al., 2007; Shacklett & Luciw, 1994). Vif protein expressed by primate lentiviruses, including human immunodeficiency virus and simian immunodeficiency virus (SIV), facilitate viral infectivity by targeting cellular cytidine deaminases of the APOBEC3 family, including APOBEC3G, for proteosomal degradation and thereby inhibit virion incorporation of these antiviral cellular proteins (Cullen, 2006; Sheehy et al., 2002). An association between feline immunodeficiency virus (FIV) Vif and a feline analogue for APOBEC3G has not been characterized at this time. However, a recent report described interactions between a feline cytidine deaminase (fe3) most related to human APOBEC3F and the feline spumavirus-encoded bet gene product (Lochelt et al., 2005). A second report revealed that replication of a non-domestic feline lentivirus (FIV-OMA) in a cell line characterized by stable expression of FIV-OMA Vif, resulted in a reduction of G-to-A mutation rates for viral genomes (Paul et al., 2007). These findings suggest that FIV Vif interacts with a feline cytidine deaminase similar to those described for primate lentivirus Vif proteins.
Although vif deletion mutants of multiple lentiviruses have proven severely attenuated after experimental infection of adult and adolescent animals, effects of vif mutation on virus replication in the neonatal host have not been reported. Infection of neonatal hosts with wild-type primate lentiviruses or FIV resulted in higher virus loads and a more rapid disease progression compared with adult infections and revealed that potential pathogenicity of a lentivirus is also complicated by the age of the host (Chakraborty, 2005; George et al., 1993; Marthas et al., 1995; Veazey et al., 2003). Furthermore, a nef-deleted SIV mutant, previously shown to be highly attenuated in adult macaques, was pathogenic and capable of producing a fatal AIDS-like disease in newborn macaques (Baba et al., 1995, 1999). In contrast, newborn macaques infected with the highly attenuated SIV clone SIVmac1A11, or with Rev-independent SIV clones, did not develop the disease (Otsyula et al., 1996; von Gegerfelt et al., 2002). These observations suggested that virus attenuation observed in adults is not always predictive of pathogenicity in the neonatal host and support the use of neonatal infection as another approach for assessing lentivirus attenuation and pathogenesis. Also, age-related differences in expression of cellular genes known to influence lentivirus replication, such as APOBEC3 family members, are not well characterized and may also influence the outcome of infection with lentiviruses attenuated by loss of accessory gene function. Accordingly, this report describes the inoculation of newborn kittens with a vif-deleted FIV provirus to further examine virus attenuation and pathogenesis associated with the loss of Vif activity. These neonatal infection studies further corroborate the importance of FIV Vif in virus replication and pathogenesis for both neonatal and adult hosts.
Our previous reports showed that inoculation of juvenile cats with a vif-deleted FIV provirus plasmid (FIV-pPPRvif) resulted in an infection detectable only by evidence of virus-specific cellular immune responses and by observation of low FIV Env antibody titres (Gupta et al., 2007; Lockridge et al., 2000). Reasons for using proviral DNA inoculation for infection of animals with this highly attenuated virus for current studies, included the severe reduction in virus particle infectivity imposed by FIV vif deletion (Lockridge et al., 1999; Paul et al., 2007; Tomonaga et al., 1992) and the possibility of sustained virus production from inoculated proviral plasmid. Furthermore, the lack of feline cell lines shown to be permissive for vif-deleted FIV isolates and the absolute restriction of FIV long terminal repeat-directed provirus expression in non-feline cell lines (Poeschla et al., 1998) provided obstacles towards the production of high titre FIV-pPPRvif particle inocula for animal infection. Lastly, our earlier studies revealed similar kinetics for induction of viraemia and antiviral antibody upon inoculation of specific-pathogen-free (SPF) juvenile cats with either FIV-pPPR provirus plasmid or with FIV-pPPR particle preparations (Dean et al., 1999; Sparger et al., 1994, 1997). Therefore, proviral DNA inoculation was chosen as the method for infection of newborn SPF kittens to examine further the importance of Vif in virus replication in vivo. The previously described FIV-pPPRvif plasmid that contains the FIV-pPPR molecular clone encoding a 375 bp deletion within the vif gene (Lockridge et al., 1999), was purified by a commercial kit (EndoFree Plasmid Mega kit; Qiagen) in preparation for inoculation of kittens. All FIV-pPPRvif and wild type (WT) FIV-pPPR plasmid preparations were tested for virus replication by electroporation of Crandell feline kidney (CRFK) cells (ATCC CCL-94) with plasmid (10 µg) followed by co-cultivation with feline peripheral blood mononuclear cells (PBMC) and assay of PBMC culture supernatants for FIVp24-Gag antigen concentrations as described previously (Gupta et al., 2006). Transfection of FIV-pPPRvif plasmid preparations revealed virus production and transient replication of low magnitude compared with replication of WT FIV-pPPR in feline PBMC (Fig. 1a). These observations were consistent with previous studies of the FIV-pPPRvif provirus plasmid (Gupta et al., 2006; Lockridge et al., 1999) and confirmed the stability of this plasmid.
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vif provirus plasmid DNA (600 µg) by the intramuscular (i.m.) route (paralumbar muscle) and a second group of three neonatal kittens (positive control group) received an i.m. inoculation with WT FIV-pPPR plasmid DNA (600 µg). A third group of three SPF newborn kittens served as uninfected controls. All inoculated kittens were monitored daily for clinical abnormalities including fever and peripheral lymphadenopathy. Blood sampling was restricted during the first 8 weeks of the study due to size and body weight of the kittens. After 8 weeks into the study, blood samples were collected every 2–6 weeks for haematological, virological and immunological assays. FIV-pPPRvif-inoculated kittens received a second i.m. injection with FIV-pPPRvif plasmid DNA (300 µg) in the rear limb quadriceps muscle at 42 weeks after primary inoculation and were evaluated for antiviral antibody and virus in peripheral blood for an additional 5 weeks, up to 47 weeks after primary inoculation. Kittens inoculated with WT FIV-pPPR DNA were removed from the study at 36 weeks after inoculation.
PBMC harvested from inoculated kittens were prepared by density-gradient centrifugation in Ficoll-Hypaque (Histopaque; Sigma-Aldrich) and cultivated for virus isolation as described previously (Dean et al., 1999). After inoculation, virus was isolated from PBMC sampled from all three kittens inoculated with WT FIV-pPPR plasmid at multiple time points, with one WT FIV-pPPR-inoculated kitten testing virus positive as early as 1 week after DNA inoculation (Table 1). In contrast, infectious virus could not be recovered from PBMC sampled from any of the five FIV-pPPRvif-inoculated kittens at all time points tested after birth. FIV proviral DNA was also not detected in PBMC preparations sampled from FIV-pPPRvif-inoculated kittens at either 42 weeks after initial inoculation or 2 weeks after a second FIV-pPPRvif inoculation (44 weeks after primary inoculation), when assayed by a quantitative real-time TaqMan PCR assay with FIV gag TaqMan probes and primer sequences and protocols described previously (Pedersen et al., 2001) (Table 1).
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) were comparable between FIV-pPPRvif-inoculated and control kittens, and a normal age-related decline in the CD4/CD8 ratios occurred in both proviral plasmid-inoculated and control groups. However, WT FIV-pPPR-inoculated kittens showed significantly lower CD4 T-cell percentages compared with control kittens for multiple time points after inoculation (Fig. 1c). Significant differences in CD4/CD8 T-cell ratios between WT FIV-pPPR-infected and control kittens were also observed, but only for selected early time points after infection (Fig. 1b). In contrast, CD8 T-cell percentages were comparable between all three groups with the exception of week 5 after infection, where the mean CD8 T-cell percentage for WT FIV-pPPR-infected kittens was greater than that measured for control kittens (data not shown). Collectively, these findings revealed severely restricted virus replication and absence of pathogenicity for FIV-pPPRvif infection of neonatal kittens when compared with WT FIV-pPPR, and reproduced observations previously reported for inoculation of juvenile cats with FIV vif mutants (Gupta et al., 2007; Inoshima et al., 1996; Lockridge et al., 2000). Interestingly, significant changes in CD4/CD8 T-cell ratios and CD4 T-cell percentages have not been reported in juvenile cats infected with the molecular clone FIV-pPPR, a cloned virus previously shown to be relatively non-pathogenic although capable of establishing significant viraemia (Dean et al., 1999; Lockridge et al., 2000; Sparger et al., 1994, 1997). Alterations in the peripheral blood CD4 T-cell population observed for WT FIV-pPPR infection of newborn kittens, further supported the use of neonatal infection studies for examination of pathogenic potential of various WT and mutant FIV isolates. This observation for WT FIV-pPPR neonatal infection contrasts with the lack of pathogenicity exhibited by FIV-pPPRvif and confirms the absolute attenuation of this FIV vif mutant.
Serum samples collected from kittens after FIV-pPPRvif inoculation were assayed for antibodies to recombinant FIV p24-Gag by a previously described ELISA (George et al., 1993). The time point for which antiviral antibody was first detectable could not be determined due to a restriction on blood sampling imposed by the size of the kittens during the first 8 weeks after birth. However, FIV p24-Gag-specific antibody was detected for all WT FIV-pPPR-inoculated kittens by 8 weeks after inoculation and ranged in titres from 1 : 102 to 1 : 105 (Table 2). Only one of five FIV-pPPRvif-inoculated kittens seroconverted, with a low antibody titre of 1 : 102 emerging at a later time point (42 weeks after inoculation). The remaining FIV-pPPRvif-inoculated and control kittens were antibody negative with titres less than 1 : 102. Antibody directed against native FIV-pPPR Env was also assayed by using a concanavalin A (Con A) antibody ELISA described previously (Lockridge et al., 2000) and was detected in WT FIV-pPPR-inoculated kittens by 7 weeks after inoculation with titres ranging from 1 : 800 to 1 : 12 800. However, very low FIV Env antibody titres observed for FIV-pPPRvif-inoculated kittens were comparable to those measured for uninfected control kittens (data not shown). Antiviral antibodies have been reported as either absent or of low titre, and infrequent in published studies characterizing in vivo pathogenesis of vif-deleted lentivirus infections (Desrosiers et al., 1998; Gabuzda et al., 1992; Gupta et al., 2007; Harmache et al., 1996; Inoshima et al., 1996; Kristbjornsdottir et al., 2004; Lockridge et al., 2000). Emergence of a low FIV Gag antibody titre (1 : 100) for one cat by 42 weeks after FIV-pPPRvif inoculation, and the subsequent boosting of this antibody titre after a second inoculation (Table 2), was interesting. This finding suggested persistent expression or virus replication of low magnitude for the inoculated mutant provirus in this particular animal, despite the absence of detectable virus. Overall, the lack of sufficient virus replication to induce a virus-specific antibody response as demonstrated for the remaining four FIV-pPPRvif-inoculated cats, further attested to the severe attenuation of a vif-deleted FIV isolate that is sustained in vivo upon inoculation of a neonatal host.
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vif replication or expression. FIV-specific T-cell proliferative responses were assayed at 32 and 36 weeks after inoculation by using a recently described flow cytometric assay where proliferation is determined by the percentage of PBMC incorporating 5-bromo-2'-deoxyuridine after stimulation with inactivated preparations of sucrose-gradient-purified virus preparations of the FIV-PPR biological isolate (Gupta et al., 2007). Proliferative responses were detected in all five FIV-pPPRvif-inoculated kittens for at least one of the two time points tested, with three of the five kittens exhibiting responses for both time points (Fig. 1d and e). Importantly, FIV-specific proliferative responses observed for FIV-pPPRvif-inoculated kittens were comparable to those measured for WT FIV-pPPR-inoculated kittens. This observation is consistent with previous reports describing evidence of virus-specific T-cell proliferative and cytotoxic responses after inoculation of juvenile and adult SPF cats with FIV-pPPRvif proviral DNA (Gupta et al., 2007; Lockridge et al., 2000), and implies sustained viral gene expression that is sufficient to produce and maintain cellular immune responses up to 36 weeks after a single inoculation at birth. Additional studies will be required to determine whether persistent provirus plasmid DNA expression or a cellular reservoir of persistent FIV-pPPRvif virus replication is responsible for these cellular responses, or for the antibody response detected for one FIV-pPPRvif-inoculated cat.
This study provided the first description of neonatal infection with a vif-deleted lentivirus and further corroborates the severe attenuation conferred on lentivirus replication in vivo by absence of the vif gene product. Given the increased susceptibility of the neonatal host to lentivirus infection and disease, experimental inoculation of newborn animals presented another approach for critical assessment of vif-deleted virus attenuation. The severely restricted virus replication and lack of pathogenicity observed for FIV-pPPRvif in newborn kittens contrasted with virus replication and pathogenicity shown for WT FIV-pPPR in this study and with the virulence reported for attenuated SIVmac239 nef deletion mutants in newborn macaques. The severity of attenuation and deficiency in cell-free infectivity previously reported for lentivirus vif mutants (Desrosiers et al., 1998; Harmache et al., 1996; Inoshima et al., 1996; Lockridge et al., 2000), suggested that these mutants might be similarly restricted for virus replication in newborn hosts. This hypothesis is now supported by findings from our current study. Future neonatal infection studies testing vif deletion mutants derived from a pathogenic lentivirus, such as SIVmac239, may be warranted to characterize fully the extent of attenuation imposed by a vif gene mutation.
Persistence of virus-specific cellular immune responses exhibited by cats after FIV-pPPRvif DNA inoculation was interesting, considering the absence of any detectable viraemia. Similar observations of strong SIV-specific T-cell proliferative responses despite weak antiviral antibody responses were also described for occult SIV and simian–human immunodeficiency virus infections in rhesus macaques induced by vaginal delivery of low dose virus inoculum (McChesney et al., 1998; Tasca et al., 2007). Future examination of mechanisms for these persistent cellular responses may be warranted to identify potential FIV-pPPRvif-permissive cell populations or reservoirs in the host and provide additional insights into Vif interactions with cellular proteins in vivo.
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ACKNOWLEDGEMENTS |
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Received 25 June 2007;
accepted 26 June 2007.
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