Adeno-Associated Vector-Delivered CRISPR/SaCas9 System Reduces Feline Leukemia Virus Production In Vitro.

Feline leukemia virus (FeLV) is a retrovirus of cats worldwide. High viral loads are associated with progressive infection and the death of the host, due to FeLV-associated disease. In contrast, low viral loads, an effective immune response, and a better clinical outcome can be observed in cats with regressive infection. We hypothesize that by lowering viral loads in progressively infected cats, using CRISPR/SaCas9-assisted gene therapy, the cat's immune system may be permitted to direct the infection towards a regressive outcome. In a step towards this goal, the present study evaluates different adeno-associated vectors (AAVs) for their competence in delivering a gene editing system into feline cells, followed by investigations of the CRISPR/SaCas9 targeting efficiency for different sites within the FeLV provirus. Nine natural AAV serotypes, two AAV hybrid strains, and Anc80L65, an in silico predicted AAV ancestor, were tested for their potential to infect different feline cell lines and feline primary cells. AAV-DJ revealed superior infection efficiency and was thus employed in subsequent transduction experiments. The introduction of double-strand breaks, using the CRISPR/SaCas9 system targeting 12 selected FeLV provirus sites, was confirmed by T7 endonuclease 1 (T7E1), as well as Tracking of Indels by Decomposition (TIDE) analysis. The highest percentage (up to 80%) of nonhomologous end-joining (NHEJ) was found in the highly conserved gag and pol regions. Subsequent transduction experiments, using AAV-DJ, confirmed indel formation and showed a significant reduction in FeLV p27 antigen for some targets. The targeting of the FeLV provirus was efficient when using the CRISPR/SaCas9 approach in vitro. Whether the observed extent of provirus targeting will be sufficient to provide progressively FeLV-infected cats with the means to overcome the infection needs to be further investigated in vivo.

[Efficacy of siRNA on feline leukemia virus replication in vitro]. / Wirksamkeit von siRNA auf die Vermehrung des felinen Leukämievirus in vitro.

Feline leukemia virus (FeLV) can lead to severe clinical signs in cats. Until now, there is no effective therapy for FeLV-infected cats. RNA interference-based antiviral therapy is a new concept. Specific small interfering RNA (siRNA) are designed complementary to the mRNA of a target region, and thus inhibit replication. Several studies have proven efficacy of siRNAs in inhibiting virus replication. The aim of this study was to evaluate the inhibitory potential of siRNAs against FeLV replication in vitro. siRNAs against the FeLV env gene and the host cell surface receptor (feTHTR1) which is used by FeLV-A for entry as well as siRNA that were not complementary to the FeLV or cat genome, were tested. Crandell feline kidney cells (CrFK cells) were transfected with FeLV-A/Glasgow-1. On day 13, infected cells were transfected with siRNAs. As control, cells were mock-transfected or treated with azidothymidine (AZT) (5 µg/ml). Culture supernatants were analyzed for FeLV RNA using quantitative real-time RT-PCR and for FeLV p27 by ELISA every 24 hours for five days. All siRNAs significantly reduced viral RNA and p27 production, starting after 48 hours. The fact that non-complementary siRNAs also inhibited virus replication may lead to the conclusion that unspecific mechanisms rather than specific binding lead to inhibition.

Feline leukaemia. ABCD guidelines on prevention and management.

OVERVIEW: Feline leukaemia virus (FeLV) is a retrovirus that may induce depression of the immune system, anaemia and/or lymphoma. Over the past 25 years, the prevalence of FeLV infection has decreased considerably, thanks both to reliable tests for the identification of viraemic carriers and to effective vaccines. INFECTION: Transmission between cats occurs mainly through friendly contacts, but also through biting. In large groups of non-vaccinated cats, around 30-40% will develop persistent viraemia, 30-40% show transient viraemia and 20-30% seroconvert. Young kittens are especially susceptible to FeLV infection. DISEASE SIGNS: The most common signs of persistent FeLV viraemia are immune suppression, anaemia and lymphoma. Less common signs are immune-mediated disease, chronic enteritis, reproductive disorders and peripheral neuropathies. Most persistently viraemic cats die within 2-3 years. DIAGNOSIS: In low-prevalence areas there may be a risk of false-positive results; a doubtful positive test result in a healthy cat should therefore be confirmed, preferably by PCR for provirus. Asymptomatic FeLV-positive cats should be retested. DISEASE MANAGEMENT: Supportive therapy and good nursing care are required. Secondary infections should be treated promptly. Cats infected with FeLV should remain indoors. Vaccination against common pathogens should be maintained. Inactivated vaccines are recommended. The virus does not survive for long outside the host. VACCINATION RECOMMENDATIONS: All cats with an uncertain FeLV status should be tested prior to vaccination. All healthy cats at potential risk of exposure should be vaccinated against FeLV. Kittens should be vaccinated at 8-9 weeks of age, with a second vaccination at 12 weeks, followed by a booster 1 year later. The ABCD suggests that, in cats older than 3-4 years of age, a booster every 2-3 years suffices, in view of the significantly lower susceptibility of older cats.

2008 American Association of Feline Practitioners' feline retrovirus management guidelines.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are among the most common infectious diseases of cats. Although vaccines are available for both viruses, identification and segregation of infected cats form the cornerstone for preventing new infections. Guidelines in this report have been developed for diagnosis, prevention, treatment, and management of FeLV and FIV infections. All cats should be tested for FeLV and FIV infections at appropriate intervals based on individual risk assessments. This includes testing at the time of acquisition, following exposure to an infected cat or a cat of unknown infection status, prior to vaccination against FeLV or FIV, prior to entering group housing, and when cats become sick. No test is 100% accurate at all times under all conditions; results should be interpreted along with the patient's health and risk factors. Retroviral tests can diagnose only infection, not clinical disease, and cats infected with FeLV or FIV may live for many years. A decision for euthanasia should never be based solely on whether or not the cat is infected. Vaccination against FeLV is highly recommended in kittens. In adult cats, antiretroviral vaccines are considered non-core and should be administered only if a risk assessment indicates they are appropriate. Few large controlled studies have been performed using antiviral or immunomodulating drugs for the treatment of naturally infected cats. More research is needed to identify best practices to improve long-term outcomes following retroviral infections in cats.

Immunomodulation therapy for feline leukemia virus infection.

Clinically ill feline leukemia virus (FeLV)-infected cats, treated with Staphylococcus protein A (SPA) or oral interferon alpha (IFN), or both, were compared with cats treated with saline (SAL). Nine cats received SPA/SAL, nine received SPA/IFN, 10 received SAL/IFN, and eight received SAL/SAL. Twelve cats survived and completed the 100-week therapy. Significantly more owners of cats treated with SPA/SAL thought their cat's health improved during treatment compared to owners of cats treated with SAL/SAL (P=0.05, pair-wise comparison) or SPA/IFN (P=0.05, pair-wise comparison). No significant differences in body weight, temperature, hematocrit, red blood cell counts, mean corpuscular hemoglobin concentration, reticulocyte counts, white blood cell or neutrophil numbers, lymphocyte concentrations, bone-marrow cytopathology, FeLV status, survival time, activity, or appetite scores were observed. No significant differences in the owners' subjective assessment of their cat's health following treatment with SAL/IFN, SPA/IFN, or SAL/SAL were seen. Therapy with SPA as a single agent results in the owners' subjective impression of improved health of their FeLV-infected cats.

Feline leukemia virus and feline immunodeficiency virus.

Ophthalmic manifestations of FeLV or FIV infection can occur in all ocular tissues and may be manifestations of direct viral effects or secondary to viral-related malignant transformation. Additionally, the manifestations of common feline ophthalmic pathogens may be more severe and poorly responsive to therapy because of the immunosuppressive effects of FeLV or FIV infection. Prompt diagnosis of underlying viral infection in cats with ophthalmic disease is paramount for accurate diagnosis and prognosis and is required for appropriate therapeutic decision making.

Treatment of feline leukemia virus-infected cats with paramunity inducer.

Two placebo-controlled double-blind trials were performed to determine the therapeutic efficacy of the paramunity inducer, Baypamun in feline leukemia virus (FeLV)-infected cats under controlled conditions. In the first study, 120 cats were involved; 60 cats were treated with Baypamun and 60 with a placebo preparation of virus-free cell culture medium. Dosage and administration of the drug over a 7-week period were performed according to the instructions given by the company. Remission of viremia occurred in 12% and 7% of the cats treated with Baypamun and placebo, respectively. This difference was not statistically significant. In the second study, 30 naturally infected cats were treated in a placebo-controlled double-blind trial. In total, 20 immunological, clinical, laboratory, and virological parameters were examined. No statistically significant differences could be demonstrated between Baypamun and placebo application. Therefore, FeLV infection was not influenced by Baypamun treatment.

Adoptive immunotherapy of feline leukemia virus infection using autologous lymph node lymphocytes.

Adoptive immunotherapy using autologous cells expanded ex vivo from lymph nodes was examined in cats infected with the retrovirus feline leukemia virus (FeLV). Cells were obtained from popliteal lymph nodes from 18 FeLV-antigen-positive cats without complications; a mean of 6.2 x 10(7) cells were obtained. Lymph node cells were cultured with 600 IU/ml interleukin-2 (IL-2) for 7 days. Cells expanded 0.8- to 11-fold (mean, 2.7; median, 2.4); were 80% +/- 8.0% CD3+, 29% +/- 8.1% CD4+, and 41% +/- 7.0% CD8+, and exhibited cytolytic activity against FeLV-transformed FL74 cells. Sixteen cats received a single intravenous infusion of 0.13 to 3.9 x 10(8) cells. Cell infusion was well tolerated; fever developed approximately 1 hour postinfusion. Clinical activity, antiviral activity, or both was observed in 10 cats. Nine cats had clinical responses with improvement in weight, activity, appearance, or a combination of these that began 2 to 4 weeks after cell infusion and that lasted for up to 13 or more months. FeLV antigen became undetectable in 4 cats. These results indicate that adoptive immunotherapy using autologous lymph node cells, activated and expanded ex vivo in short-term cultures with low concentrations of IL-2, can modulate the course of a retroviral infection.

[Placebo-controlled double-blind study on the efficacy of a paramunity inducer in the treatment of naturally FeLV-infected cats]. / Plazebokontrollierte Doppelblindstudie über die Wirksamkeit eines Paramunitätsinducers bei natürlich FeLV-infizierten Katzen.

The study involved 120 cats, of which 60 were treated with Baypamun HK and 60 cats received a preparation of virus free cell culture medium. Therapeutic efficacy was determined by monitoring the general status of health, body weight, skin, lymph nodes, oral cavity, the presence of the p27 antigen and the FeLV p27 antigen concentration in serum. No statistically significant differences between both groups could be demonstrated neither for clinical nor for virologic parameters. Remission of the viremia occurred in 11.7% of the cats treated with Baypamun HK and in 6.7% of the cats treated with placebo. The FeLV p27 antigen level decreased by an average of 7.2% with Baypamun HK and by an average of 5.1% with placebo.

Caring for the retrovirus infected cat.

No commercial vaccine [correction of vacine] exists for feline immunodeficiency virus (FIV), and although feline leukemia virus (FeLV) vaccines are available, they are neither 100% effective nor used in all cats. These realities clearly indicate the veterinarian will be required to treat either FeLV- or FIV-positive cats for some time to come. The management of FIV- or FeLV-positive cats may require supportive therapies as well as virus-specific therapies such as zidovudine (AZT; Retrovir, Burroughs Wellcome, Research Triangle Park, NC).

Opportunistic infections associated with retroviral infections in cats.

Naturally occurring infection of cats with feline leukemia virus and feline immunodeficiency virus is common. A wide variety of clinical manifestations occur in retrovirus-infected cats including gastrointestinal tract disease, respiratory tract disease, central nervous system disease, ophthalmic disease, hepatic disease, urogenital tract disease, dermatologic disease, hematologic disease, and musculoskeletal disease. Clinical signs are often directly attributable to the primary viral infection, but because both feline leukemia virus and feline immunodeficiency virus can induce immunodeficiency, opportunistic secondary infections may cause the clinical manifestations of disease in some cats. Diagnosis, treatment, and zoonotic potential of the common opportunistic agents associated with feline retroviral infections are reviewed.

Reversal of feline leukemia virus infection by adoptive transfer of activated T lymphocytes, interferon alpha, and zidovudine.

Previous experimental studies utilizing human recombinant interferon-alpha-2b (IFNalpha-2b) alone or with zidovudine (AZT) to treat established feline leukemia virus (FeLV) infection resulted in a significant reduction in circulating virus throughout a 49-day treatment period. However, the anti-FeLV effect of IFNalpha was limited by the production of IFNalpha-neutralizing antibodies detected 7 weeks after the start of treatment. AZT without IFNalpha had no effect on circulating virus load. To examine the hypothesis that combination chemoimmunotherapy might induce the clearance of FeLV infection, persistently infected cats were infused with activated lymphocytes, IFNalpha, and AZT 12 weeks after infection with FeLV. Recipient cats received weekly infusions of 1.46 x 10(8) lymphocytes activated in vitro with lectin/IL-2 comprised of 98% T cells and an even distribution of CD4+ and CD8+ lymphocytes. FeLV infection was cleared in 4 of 9 cats receiving combined therapy after four adoptive cell transfers. These cats remained negative for circulating virus during a 63-day treatment period (17 adoptive cell transfers) despite the production of anti-IFNalpha-neutralizing antibodies. Sequential development of virus-neutralizing and virus envelope antibody titers were detected in those cats which cleared retroviremia, an antiviral response that was absent in untreated control animals or nonresponders. Three of four responder cata remained negative for FeLV 95 days after treatment was discontinued. Treatment of cats with lymphocytes without chemotherapy failed to influence the course of FeLV infection. These results suggest that combined treatment using IFNalpha and adoptive lymphocyte transfer served to reconstitute antiviral humoral immunity, counteract immunosuppression, and induce the reversal of retroviremia.

Husbandry practices for cats infected with feline leukemia virus or feline immunodeficiency virus.

Cats that are persistently infected with FeLV or feline immunodeficiency virus but are not manifesting clinical signs of disease are at risk for developing a wide variety of immunosuppressive, degenerative, or neoplastic diseases. Infected cats should be isolated to prevent transmission of virus to healthy cats, and to protect infected cats from exposure to pathogens that can cause life-threatening secondary infections. Iatrogenic transmission of virus from infected cats in isolation to healthy cats may be reduced by strict adherence to handling, sanitation, and disinfection procedures. Husbandry practices that may delay the complications of infection include regular vaccination, provision of high-quality diets, reduction of stress, control of endoparasites and ectoparasites, and early and aggressive treatment of clinical signs of disease.