Chicken Anemia Virus and Immunosuppression: Impact on Marek´s Disease Vaccine ProtectionSummary Subclinical immunosuppression caused by chicken infectious anemia virus (CIAV) is an important contributing factor to Marek’s disease (MD) vaccine breaks. Infection with CIAV results in reduced T helper and cytotoxic T lymphocyte activity affecting antibody and cell-mediated immune responses. CIAV infection is controlled by the development of virus-neutralizing antibodies, which can be compromised by poorly controlled infectious bursal disease virus (IBDV) infection. MD virus (MDV), especially the very virulent (vv+) strains, is also highly immunosuppressive. When field strains of MDV infect properly vaccinated birds or reactivate from latency, memory CTL will be activated to control virus replication. These CTL are also dividing thus providing target cells for CIAV replication. In conclusion, when CIAV is actively replicating during infection with or reactivation of MDV, CTL responses are suboptimal and MDV infection is poorly controlled leading to vaccine breaks. INTRODUCTION Although MD is in general well controlled by vaccination in ovo or at one d of age, MD remains a concern for several reasons. First of all, vaccination practices are often suboptimal resulting in some vaccine breaks. Proper use of standard operating procedures at the hatchery remains essential for optimal protection and has been the topic of many presentations. The short-term financial gain by using vaccines diluted beyond the recommendations by the manufacturer results in suboptimal protection when very virulent (vv) or vv+ strains of MDV are present (4). The second reason is the continuous evolution of MDV. Over the last 100 years MDV has increased significantly in virulence (10). The first increase in virulence occurred in the mid 1950’s when the poultry industry changed from a rather extensive to a more intensive production system. Subsequent increases in virulence are, at least in part, caused by the fact that none of the vaccines prevent infection with field strains thus allowing for the development of escape mutants (1). In addition, Atkins et al. (1) suggested that the reduction in age of broilers to processing also favors an increase in virulence. Unfortunately, there are no good options to change these developments. Without vaccination, losses would be staggering in breeder and layer flocks and in many countries in broilers as well. Thorough cleaning of broiler houses after each cycle may alleviate the need to vaccinate as is the case in some countries, but this will be impractical in the USA and Mexico. A third important factor is immunosuppression especially by chicken infectious anemia virus (CIAV), which is difficult to control in commercial production systems. In this review I will briefly discuss the pathogenesis of CIAV and its impact on immune responses, MD vaccine-induced immune responses, and how CIAV can influence MD vaccine-induced immune responses. With few exceptions only references for book chapters and review papers are used for these three sections. PATHOGENESIS OF CHICKEN INFECTIOUS ANEMIA VIRUS CIAV, currently the only member of the Gyrovirinae of the Circoviridae, is characterized by its small size (±25 nm), single-stranded, circular, covalently closed, negative sense DNA genome of 2,298 nt, and very important from a practical point of view the extreme resistance of CIAV to many commercial disinfectants (7, 9). The genome codes for only three proteins: VP1 (the capsid protein), VP2 (essential for the proper folding of VP1) and VP3, which is also known as apoptin. VP3 is essential for virus replication and mutation of the start codon will prevent virus replication. VP3 is also important because it causes apoptosis of infected cells. The replication of the viral genome requires the formation of double-stranded (ds)DNA, which resembles in some respects a mini-chromosome or a bacterial plasmid. Because CIAV does not code for the necessary enzymes to generate new DNA, and thus infectious virus particles, it needs to infect dividing cells using the cellular enzymes to generate viral DNA. The dividing cells which are susceptible to infection with CIAV are the hemocytoblasts, the precursor cells for erythrocytes, heterophils and thrombocytes, in the bone marrow, thymocytes and T cells. Destruction of the hemocytoblasts by CIAV results in lower hematocrit values, decreased phagocytosis of bacteria by a lack of heterophils and thrombocytes, and increased hemorrhages. Infection of the thymocyte series results in a loss of thymocytes (thymus atrophy), T helper (Th) and cytotoxic T lymphocytes (CTL). The loss of Th lymphocytes and CTL impacts negatively the antibody and cell-mediated immune responses. Virus-neutralizing (VN) antibodies develop within six wk post infection (pi) eliminating virus replication. However, CIAV can remain present in gonads and lymphocytes probably as dsDNA fulfilling the characteristics of latency (7). Latent CIAV can be transmitted vertically and be reactivated. Infection with CIAV only causes clinical disease if infection occurs during the first one to ten d of age in maternal antibody-negative chickens. However older chickens can develop clinical disease when humoral antibody responses are severely compromised for example after infection with vv infectious bursal disease virus (IBDV). Control of vvIBDV using appropriate vaccines without causing damage to the bursa of Fabricius is therefore an important component for the control of CIAV infections. MAREK’S DISEASE VACCINE-INDUCED IMMUNITY Infection of naïve chickens with MDV causes first a lytic infection of B lymphocytes followed by a lytic infection of mostly CD4+ T cells. The lytic phase of infection can cause severe atrophy of the thymus and bursa of Fabricius resulting in immunosuppression. Latent infections are established in CD4+ T cells starting around seven d pi but infection with vv+ strains may cause permanent damage to the primary lymphoid organs and early mortality. Latency can be permanent or temporarily depending on the genetic resistance and immunocompetence of the birds and the virulence of the MDV strain. Ultimately, MDV-positive CD4+ cells may transform in which case tumors develop. To protect against MD, chickens are vaccinated in the USA at 18 d of embryonation (broilers) or directly after hatching (layers and breeders). The latter two groups of birds receive sometimes a second vaccination between 1 – 14 d of age. If a second vaccination is given, it has to be done within the first one to two d of age before chickens are exposed to field virus. Vaccination induces both innate and acquired immune responses. The former include the production of nitric oxide and interferons as well as the activation of natural killer cells which are important to reduce early MDV infections. Innate responses are short-lived, lack memory, and are therefore only important during the first 7 – 10 d pi. However, innate responses are of crucial importance for the development of acquired immunity. Antibodies play only a minor role in protective immunity because MDV infection is strictly cell-associated. In contrast to antibodies, CTL responses are a key component of vaccine-induced acquired immunity. Protective immunity is primarily antiviral reducing but not preventing replication of field virus. The importance of antitumor immunity is controversial and immune responses to tumor cells may be directed to viral antigens rather than true tumor antigens. (8). IMPACT OF CIAV INFECTION ON MAREK’S DISEASE CIAV infection in maternal antibody-positive chickens typically occurs once maternal antibodies have weaned and flocks typically seroconvert between 4 – 10 wk of age. During this time birds may also become infected with MDV field strains. CTL responses are the key component to control virus replication and memory CTL against MDV antigens will be rapidly activated and start dividing thus presenting target cells for CIAV replication. MD vaccine breaks have been linked directly or indirectly to the presence of CIAV infection in several instances (2, 3). Similarly, CIAV infection has also been implicated in infectious bronchitis breaks (5). The effect of CIAV on CTL was clearly shown by Markowski-Grimsrud and Schat (6) using reticuloendotheliosis virus (REV) as a model. Chickens hatched from antibody-positive and -negative hens were infected at four wk of age with CIAV with or without exposure to REV at the same time. At seven d pi CIAV replication was measured by quantitative (q)PCR and qRT-PCR and CTL responses to REV-transformed lymphocytes was measured by chromium release assays (CRA). In the case of maternal antibody-positive chickens, qPCR and qRT-PCR showed lack of CIAV replication and a strong CTL response to REV. Residual maternal antibodies were apparently still present at four wk of age, even while the Iddex ELISA was negative. In contrast, maternal antibody-negative chicks showed high levels of CIAV DNA and RNA, the latter indicating active virus replication. The CTL response to REV was significantly reduced in these birds. CONCLUSIONS CIAV is an important pathogen causing subclinical immunosuppression and can be an important co-factor in vaccine breaks against MD and may other diseases. Development of vaccines to protect chickens to CIAV infection early in life will be an important addition to disease control programs. REFERENCES 1. Atkins, K. E., A. F. Read, N. J. Savill, K. G. Renz, A. F. Islam, S. W. Walkden-Brown, and M. E. Woolhouse. Vaccination and reduced cohort duration can drive virulence evolution: Marek's disease virus and industrialized agriculture. Evolution 67:851-860. 2013. 2. Davidson, I., M. Kedem, H. Borochovitz, N. Kass, G. Ayali, E. Hamzani, B. Perelman, B. Smith, and S. Perk. Chicken infectious anemia virus infection in Israeli commercial flocks: virus amplification, clinical signs, performance, and antibody status. Avian Dis. 48:108–118. 2004. 3. Fehler, F., and C. Winter. 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