High Viremia Research Articles

Th2 Polarization in Peripheral Blood Mononuclear Cells from Human Immunodeficiency Virus (HIV)-Infected Subjects, as Activated by HIV Virus-Like Particles

We have recently shown that human immunodeficiency virus type 1 (HIV-1) Pr55(gag) virus-like particles (HIV-VLPs), produced in a baculovirus expression system and presenting a gp120 molecule from a Ugandan HIV-1 isolate of clade A, induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4(+) T cells in an ex vivo immunization assay. In the present study, we show that similar data can be obtained directly with fresh peripheral blood mononuclear cells (PBMCs), and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14(+) cell populations to an immunogenic stimulus. Some HIV-1-seropositive subjects, however, show a complete lack of maturation induced by HIV-VLPs in CD14(+) circulating cells, which does not consistently correlate with an advanced status of HIV-1 infection. The established Th2 polarization in both HIV-seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for therapeutic effectiveness in HIV-1-infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of "responsive" vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.

Understanding the contribution of cellular immunity to dengue disease pathogenesis

Dengue viruses (DENV) are the mosquito-borne viruses of greatest global public health importance. DENV circulate as four serotypes with significant immunologic cross-reactivity that does not provide protection from secondary infection with heterologous serotypes. The strong association of severe dengue illness, dengue hemorrhagic fever (DHF), with heterologous secondary infection and high cytokine levels has led to a prevailing view that DHF is immunologically mediated. In vitro studies of DENV-specific T lymphocytes, clinical studies of acute DENV infection, and immunologic studies in mouse models have provided evidence that in heterologous secondary DENV infection, there is preferential activation of memory T lymphocytes with lower avidity for the infecting virus ('original antigenic sin') resulting in altered T-cell functional responses. In the setting of host genetic predisposition and high level viremia, with resulting high antigenic burden, we postulate that a skewed T-cell cytokine response leads to plasma leakage in DHF. A better understanding of the immune responses associated with increased or decreased risk for DHF will be of immense value for the clinical studies of candidate multivalent DENV vaccines anticipated to take place in the next several years.

Lysis of Endogenously Infected CD4+ T Cell Blasts by rIL-2 Activated Autologous Natural Killer Cells from HIV-Infected Viremic Individuals

Understanding the cellular mechanisms that ensure an appropriate innate immune response against viral pathogens is an important challenge of biomedical research. In vitro studies have shown that natural killer (NK) cells purified from healthy donors can kill heterologous cell lines or autologous CD4+ T cell blasts exogenously infected with several strains of HIV-1. However, it is not known whether the deleterious effects of high HIV-1 viremia interferes with the NK cell-mediated cytolysis of autologous, endogenously HIV-1-infected CD4+ T cells. Here, we stimulate primary CD4+ T cells, purified ex vivo from HIV-1-infected viremic patients, with PHA and rIL2 (with or without rIL-7). This experimental procedure allows for the significant expansion and isolation of endogenously infected CD4+ T cell blasts detected by intracellular staining of p24 HIV-1 core antigen. We show that, subsequent to the selective down-modulation of MHC class-I (MHC-I) molecules, HIV-1-infected p24pos blasts become partially susceptible to lysis by rIL-2-activated NK cells, while uninfected p24neg blasts are spared from killing. This NK cell-mediated killing occurs mainly through the NKG2D activation pathway. However, the degree of NK cell cytolytic activity against autologous, endogenously HIV-1-infected CD4+ T cell blasts that down-modulate HLA-A and –B alleles and against heterologous MHC-Ineg cell lines is particularly low. This phenomenon is associated with the defective surface expression and engagement of natural cytotoxicity receptors (NCRs) and with the high frequency of the anergic CD56neg/CD16pos subsets of highly dysfunctional NK cells from HIV-1-infected viremic patients. Collectively, our data demonstrate that the chronic viral replication of HIV-1 in infected individuals results in several phenotypic and functional aberrancies that interfere with the NK cell-mediated killing of autologous p24pos blasts derived from primary T cells.

Are HCV genotypes 2 and 3 the same or different?

Because hepatitis C virus genotypes 2 and 3 are both responsive to antiviral treatment, they have been regarded as similar. However, recent evidence has shown there may be differences between these two genotypes with regard to their clinical features and possibly responses to combined interferon and ribavirin therapy. This article reviews the recent literature analyzing these potential differences, particularly reports suggesting a relationship between high baseline viremia levels and lower treatment response in genotype 3. However, as demonstrated in recent studies considering rapid virologic response (RVR) as a criterion for shortening treatment from the standard 24 weeks to 12 or 16 weeks in patients with genotype 2 or 3, the reason for lower response in genotype 3 versus genotype 2 patients may be a lower response rate in patients without an RVR. As a consequence, treatment longer than the currently recommended 24 weeks may be required for patients with genotype 3 but not for those with genotype 2 in the absence of an RVR. Whether this lower sensitivity to treatment in a subgroup of patients with genotype 3 may be attributed to viral heterogeneity or other associated cofactors is not yet known.

Public Health Dilemmas Concerning a 2-year old Hepatitis-B Carrier – Response

The public health physician is concerned about the possibility that, at a nursery, this boy would have close contact with other children and might infect them with hepatitis B virus (HBV). HBV infection in children is often asymptomatic, and acute disease with severe complications is rare. However, children often become chronic carriers after infection, which may lead to cirrhosis and liver cancer. HBV carriers also remain life long sources of infection to others and hence contribute to the spread of disease. HBV is mainly spread via blood and semen. Transmission through saliva, though unlikely, is certainly possible, especially where the boy shows a high HBV viremia [1]. The risk of transmission in day care is low, but has been reported and confirmed by genotyping [2–6]. The theoretical routes of transmission are direct through small wounds and, more important, by child bites. Indirect transmission is also possible through the sharing of utensils including toothbrushes, and, to a lesser extent, the sharing of toys that children may put in their mouth. Only very few countries with low incidence and prevalence of HBV infection, have not yet included hepatitis B in their universal childhood vaccination program. Universal vaccination prevents (most) day care transmission. This case occurred in TheNetherlands, and as The Netherlands does not have a universal HBVvaccination program, one could assume that most of the children in this nursery would be susceptible to HBV infection. However, there seems to be no scientific consensus that the vaccine does offer full protection against the G145R-HBV-mutant. While animal studies suggest that the vaccine does protect against this mutant [7] there remains ongoing uncertainty regarding the effectiveness of the vaccine [8]. The case description presents several policy options: preventing the child from attending the nursery; allowing him to go but with strict hygienic measures at the nursery; and offering vaccination to all other children at the nursery. All these possibilities may be detrimental to the child’s welfare because he will be treated differently by care-givers and other parents. And even if day care staff and other parents are not informed which child is the source of risk, it may be difficult to keep it a secret. This would be more so if care-givers are informed and subsequently are more attentive to possible risk-contacts of the boy. Other parents may find out about the identity of the child and try to avoid contact between him and their own children. Moreover, the theoretical possibility that this child may be infected with a virus against which there is less protection could easily lead them to overestimate the risks of infection. Bioethical Inquiry (2008) 5:87–89 DOI 10.1007/s11673-008-9088-3

Primary HIV infection: to treat or not to treat?

The aim of this article is to review new data on the use of antiretroviral therapy in primary HIV infection. The concept of 'hit HIV hard and early' supporting the use of antiretroviral therapy to treat the early stages of HIV infection has been revisited. Recent demonstration of massive, rapid and largely irreversible HIV-mediated destruction of memory CD4+ T cells predominantly occurring in the gut suggests that primary HIV infection may be the only time that intervention could confer lasting immunological benefit. There are no data, however, from randomized controlled trials supporting the use of antiretroviral therapy in primary HIV infection, although a number of observational studies have demonstrated limited, transient improvements in measured immunological outcomes. The first randomized controlled trials powered to address whether antiretroviral therapy of a limited duration in primary HIV infection can influence long-term CD4 decline (SPARTAC) is now fully recruited but will report in 2-3 years. In addition, given that individuals with primary HIV infection contribute disproportionately towards onward HIV transmission, there may be an additional public health impetus for earlier diagnosis and intervention in primary HIV infection. In the absence of randomized controlled trials data demonstrating clinical benefit of antiretroviral therapy intervention in primary HIV infection, clinical guidelines remain unclear. Intervention seems a logical strategy to counter the high viraemia, enhanced onward transmissibility, and immunological destruction which occurs during primary HIV infection. The nature, duration and timing of that intervention after HIV acquisition remains unknown.

Molecular Epidemiology and Clinical Significance of Hepatitis B Virus Genotypes, Core Promoter and Precore Mutations in Eastern India

Objectives: This unmatched case-control study aimed at determining the molecular epidemiology and clinical significance of HBV genotypes, core promoter (CP) and precore (PC) mutations in Eastern India. Methods: Serological, biochemical and molecular assays were used to examine antigens, ALT, genotypes, mutations and viremia among 106 inactive carriers and 183 chronic liver disease (CLD) patients. Results: Male gender (p < 0.001), HBeAg positivity (p = 0.050), high ALT (p < 0.001), high viremia (p < 0.001), CP mutations (p < 0.001), and genotypes A (p < 0.001) and C (p = 0.027) were significantly associated with CLD. Subjects infected with genotypes A and C had significantly higher prevalence of BCP mutations (p < 0.001), and low incidence of PC mutation (p < 0.001 and p = 0.047, respectively). Prevalence of genotype D was significantly higher among subjects with history of familial/childhood jaundice, while genotypes A and C were frequent among subjects with possible percutaneous exposure. Conclusions: Significant differences in risk factors and disease manifestation do exist among patients infected with different HBV genotypes. Genotypes A and C are frequently found among chronic liver disease patients, while genotype D is associated with inactive HBeAg-negative infections. This evaluation of clinical relevance of HBV genotypes, mutations and risk factors may be useful in disease prognosis, management and prevention strategies.

614 SERUM GROWTH FACTORS AND APOPTOSIS MARKERS REFLECT INFLAMMATORY ACTIVITY IN CHRONIC HEPATITIS C

R-848 (TLR7/8L), c. serum from a patient chronically infected by HBV (as control), with high viraemia levels (−9 log10), d. serum from a patient chronically infected by HCV, with high viraemia levels (−7 log10), e. supernatant from 2215 hepatocytes (HBV progeny), f. various concentrations of ribavirin. Pulsing with all these stimuli took place either with or without the presence of CD40L-expressing cells as additional pDC activation stimulus. The aim was to explore the effect of the stimuli used, to the pDC Th1skewing capacity and type I IFN production (i.e. secretion of IFN-a, IL-6 and IL-12p40, in cell-culture supernatants, measured with commercially available ELISA kits) and to compare the effect of known TLR ligands (CpG ODN and R-848) to the effect of HCV and also the effect of a component of the currently established antiviral treatment (Ribavirin). Results: Our data demonstrate: a. stimulation of IL-6 production by CpG ODN and HBV serum/2215 S/N at comparable levels, stimulation of IL-6 production by R-848 at levels 3−4 times higher than the ones elicited by HCV serum, b. IL-12p40 production stimulation by R-848 at levels 3−4 times higher than the ones observed after incubation with HCV serum and also very high levels of IL-12p40 after stimulation with Ribavirin, c. IFNa production stimulated by CpG ODN and R-848 at levels comparable to the ones observed with HBV and HCV serum pulsing, respectively. Conclusions: It appears that: (a) the Th-1 skewing capacity of pDC is partially affected by HCV infection, (b) Ribavirin possibly exerts an immunomodulatory role by triggering IL-12 production (Th1 skewing).

The Trap

The Centers for Disease Control and Prevention, national public health agencies, and an expert committee of the European Commission have recently published updated guidelines on postexposure prophylaxis (PEP) of occupational human immunodeficiency virus (HIV) exposure. 1,2 Management of occupational HIV exposure relies heavily on HIV antibody testing of the source patient. However, patients with acute HIV infection may be antibody negative yet particularly contagious because of high viremia. We report 2 cases involving health care workers who were exposed to patients with acute primary HIV infection (PHI). The patients were in the window period of seroconversion, during which detection of HIV p24 antigen provided the definitive indication for PEP. We also review the literature on the topic.

Molecular diagnostics for the detection of human flavivirus infections

Flaviviruses constitute a genus of viruses that are important etiologic agents of human disease, causing clinical disease ranging from fever to severe manifestations, such as encephalitis and hemorrhagic fever. Serology is presently the most frequently used means of diagnosing flavivirus infections. However, other diagnostic tests may be employed, such as molecular detection, virus isolation and antigen-capture procedures. The applicability of the latter three diagnostic procedures can be expected to vary depending upon the infecting flavivirus, as some flaviviruses, such as dengue, display high and long-term viremias, whereas other flaviviruses produce no, or barely detectable, viremias. Molecular diagnostic techniques have been successfully applied to the diagnosis of flavivirus infections and have the advantage of rapidity, sensitivity and specific identification of the infecting virus. However, it is important to ensure that the right detection tools are employed (for example, appropriate primers and probes to detect the specific virus) and that the laboratory maintains a high proficiency in their testing procedures. Some of the studies that have been employed in the diagnosis of flavivirus infections are reviewed in this article. It seems that there is the potential to develop testing algorithms that successfully employ molecular diagnostics alone or in conjunction with other laboratory techniques for the diagnosis of acute human flavivirus infections.

Case Report: Hepatitis C Virus Activation in HIV-Infected Patients Initiating Highly Active Antiretroviral Therapy

We describe repeated episodes of hepatitis C (HCV) activation associated with initiation of highly active antiretroviral therapy (HAART) in two HIV/HCV coinfected individuals with undetectable serum HCV RNA. Both patients developed high HCV viremia (>1 million IU/mL) and elevations in aminotransferases >10 times upper limit of normal) within 4 months of starting HAART. This is the first report of clinically significant HCV activation in HCV-seropositive patients with initially undetectable HCV viremia. These observations suggest that flares of hepatitis C in the setting of the immune reconstitution inflammatory syndrome can occur even in those patients who have undetectable serum HCV levels prior to HAART initiation.

Humanized NOD/SCID/IL2RγnullMice Transplanted with Hematopoietic Stem Cells under Nonmyeloablative Conditions Show Prolonged Life Spans and Allow Detailed Analysis of Human Immunodeficiency Virus Type 1 Pathogenesis

In a previous study, we demonstrated that humanized NOD/SCID/IL2Rgamma(null) (hNOG) mice constructed with human hematopoietic stem cells (HSCs) allow efficient human immunodeficiency virus type 1 (HIV-1) infection. However, HIV-1 infection could be monitored for only 43 days in the animals due to their short life spans. By transplanting HSCs without any myeloablation methods, the mice successfully survived longer than 300 days with stable engraftment of human cells. The mice showed high viremia state for more than the 3 months examined, with systemic HIV-1 infection and gradual decrease of CD4+ T cells analogous to that in humans. These capacities of the hNOG mice are very attractive for modeling mechanisms of AIDS progression and therapeutic strategy.

PD-1 upregulation is associated with HBV-specific T cell dysfunction in chronic hepatitis B patients

Programmed death-1 (PD-1) is demonstrated to have an increased expression on antigen-specific T cells during chronic virus infections, and the blockage of PD-1/PD-ligand (PD-L1) pathway could restore the function of exhausted T cells. We measured the PD-1 expression levels on HBV-specific CD8 T cells and investigated the role of PD-1/PD-L1 pathway in T-cell responses of patients with different HBV infection statuses. Compared to the patients with convalescent acute hepatitis B, PD-1 expression on total CD8 T cells from chronic hepatitis B (CHB) patients was significantly upregulated, especially on the HBV pentamer-positive CD8 T cells. And PD-L1, but not PD-L2, was also significantly upregulated on PBMC from CHB patients. In CHB patients, HBV-specific T cells and cellular proliferation could be observed under the recombinant HBV-Ag stimulation in vitro, and blockade of PD-1 pathway significantly enhanced the IFN-gamma production and cellular proliferation of PBMC. Furthermore, PD-1 expression level on HBV-pentamers positive CD8 T cells was positively associated with plasma viral load in CHB patients. Thus, PD-1 upregulation on HBV-specific CD8 T cells is engaged in the dysfunction of T cells and high viraemia in CHB patients, and the antiviral T-cell responses could be improved by the blockade of this inhibitory PD-1/PD-L1 pathway.

Preventing hepatitis C virus infection in injection drug users: risk reduction is not enough

Preventing hepatitis C virus infection in injection drug users: risk reduction is not enough

Editorial: Scale‐up of antiretroviral therapy in sub‐Saharan Africa – priorities for public health research

1 London School of Hygiene and Tropical Medicine, London, UK 2 Uganda Virus Research Institute, Entebbe, Uganda 3 Department for International Development, Kampala, Uganda 4 The AIDS Support Organisation, Kampala, Uganda 5 Wakiso District Council, Entebbe, Uganda 6 HIV Division, World Health Organisation, Geneva, Switzerland 7 Centre for Sexual Health & HIV Research, University College London, London, UK

Regulatory T-Cell Markers, Indoleamine 2,3-Dioxygenase, and Virus Levels in Spleen and Gut during Progressive Simian Immunodeficiency Virus Infection

High levels of viral replication occur in gut-associated lymphoid tissue (GALT) and other lymphoid tissues (LT) since the early phase of human/simian immunodeficiency virus (HIV/SIV) infection. Regulatory T cells (T(reg)), a subset of immunosuppressive T cells expressing CTLA-4 and the FoxP3 transcription factor, accumulate in LT during HIV/SIV infection. Here we show that FoxP3 and CTLA-4 mRNA are increased in leukocytes from the spleens, lymph nodes (LN), and mucosal sites of chronically SIV-infected macaques with high viremia (SIV(HI)) compared to animals with low viremia (SIV(LO)). FoxP3 and CTLA-4 correlated with SIV RNA levels in tissues; SIV virus levels in the spleen, inguinal LN, mesenteric LN, colon, and jejunum directly correlated with the plasma virus level. Importantly, CTLA-4 and FoxP3 mRNA were predominantly increased in the CD25(-) subpopulation of leukocytes from SIV(HI), further challenging the classical definition of T(reg) as CD4(+) CD25(+) T cells. Similar to CTLA-4 and FoxP3, expression of indoleamine 2,3-dioxygenase (IDO), an immunosuppressive enzyme induced by T(reg) in antigen-presenting cells, was increased in the spleens, mesenteric LN, colons, and jejuna from SIV(HI) compared to SIV(LO) and directly correlated to SIV RNA in the same tissues. Accordingly, plasma kynurenine/tryptophan, a marker for IDO enzymatic activity, was significantly higher in SIV(HI) compared to SIV(LO) and correlated with plasma viral levels. Increased T(reg) and IDO in LT of SIV-infected macaques may be the consequence of increased tissue inflammation and/or may favor virus replication during the chronic phase of SIV infection.

Significance of arginine 20 in the 2A protease for swine vesicular disease virus pathogenicity

Pathogenic and attenuated strains of swine vesicular disease virus (SVDV), an enterovirus, have been characterized previously and, by using chimeric infectious cDNA clones, the key determinants of pathogenicity in pigs have been mapped to the coding region for 1D-2A. Within this region, residue 20 of the 2A protease is particularly significant. Inoculation of pigs with mutant viruses containing single amino acid substitutions at this residue leads to the appearance of revertants, often containing an arginine at this position encoded by an AGA codon, one of six codons for this residue. The properties in pigs of two chimeric viruses, each with an arginine residue at this position but encoded by different codons, have been investigated in parallel with the parental pathogenic and attenuated strains. Presence of the arginine residue, but not of the AGA codon, is essential for induction of high viraemia and appearance of significant disease.

Induction of potent local cellular immunity with low dose X4 SHIV SF33A vaginal exposure

Intravaginal inoculation of rhesus macaques with varying doses of the CXCR4 (X4)-tropic SHIV SF33A isolate revealed a threshold inoculum for establishment of systemic virus infection and a dose dependency in overall viral burden and CD4+ T cell depletion. While exposure to inoculum size of 1000 or greater 50% tissue infectious dose (TCID 50) resulted in high viremia and precipitous CD4+ T cell loss, occult infection was observed in seven of eight macaques exposed to 500 TCID 50 of the same virus. The latter was characterized by intermittent detection of low level virus with no evidence of seroconversion or CD4+ T cell decline, but with signs of an ongoing antiviral T cell immune response. Upon vaginal re-challenge with the same limiting dose 11–12 weeks after the first, classic pathogenic X4 SHIV SF33A infection was established in four of the seven previously exposed seronegative macaques, implying enhanced susceptibility to systemic infection with prior exposure. Pre-existing peripheral SIV gag-specific CD4+ T cells were more readily demonstrable in macaques that became systemically infected following re-exposure than those that were not. In contrast, early presence of circulating polyfunctional cytokine secreting CD8+ T cells or strong virus-specific proliferative responses in draining lymph nodes and in the gut associated lymphoid tissue (GALT) following the first exposure was associated with protection from systemic re-infection. These studies identify the gut and lymphoid tissues proximal to the genital tract as sites of robust CD8 T lymphocyte responses that contribute to containment of virus spread following vaginal transmission.

Immunization with hepatitis C virus-like particles results in control of hepatitis C virus infection in chimpanzees.

Recombinant hepatitis C virus (HCV)-like particles (HCV-LPs) containing HCV structural proteins (core, E1, and E2) produced in insect cells resemble the putative HCV virions and are capable of inducing strong and broad humoral and cellular immune responses in mice and baboons. Here, we present evidence on the immunogenicity and induction of protective immunity by HCV-LPs in chimpanzees. Chimpanzees (two in each group), were immunized with HCV-LPs or HCV-LPs plus AS01B adjuvant. After immunizations, all animals developed an HCV-specific immune response including IFN-gamma(+), IL-2(+), CD4(+), and CD8(+) T cell and proliferative lymphocyte responses against core, E1, and E2. Upon challenge with an infectious HCV inoculum, one chimpanzee developed transient viremia with low HCV RNA titers (10(3) to 10(4) copies per ml) in the third and fourth weeks after the challenge. The three other chimpanzees became infected with higher levels of viremia (10(4) to 10(5) copies per ml), but their viral levels became unquantifiable (<10(3) copies per ml) 10 weeks after the challenge. After the HCV challenge, all four chimpanzees demonstrated a significant increase in peripheral and intrahepatic T cell and proliferative responses against the HCV structural proteins. These T cell responses coincided with the fall in HCV RNA levels. Four naïve chimpanzees were infected with the same HCV inoculum, and three developed persistent infection with higher viremia in the range of 10(5) to 10(6) copies per ml. Our study suggests that HCV-LP immunization induces HCV-specific cellular immune responses that can control HCV challenge in the chimpanzee model.

Differential immunity in pigs with high and low responses to porcine reproductive and respiratory syndrome virus infection1,2

One hundred Hampshire x Duroc cross-bred pigs (HD) and 100 NE Index line (I) pigs were infected with porcine reproductive and respiratory syndrome (PRRS) virus and evaluated for resistance/susceptibility. Controls (100/line) were uninfected littermates to the infected pigs. Viremia, change in weight (WTdelta), and rectal temperature at 0, 4, 7, and 14 d postinfection were recorded. Lung, bronchial lymph node (BLN), and blood tissue were collected at necropsy (14 d postinfection). The first principal component from principal component analyses of all variables was used to rank the pigs for phenotypic response to PRRS virus. Low responders (low PRRS burden) had high WTdelta, low viremia, and few lung lesions; high responders (high PRRS burden) had low WTdelta, high viremia, and many lesions. The RNA was extracted from lung and BLN tissue of the 7 highest and 7 lowest responders per line and from each of their littermates. Expression of 11 innate and T helper 1 immune markers was evaluated with cDNA in a 2 x 2 x 2 factorial design. Significant upregulation in lung, lymph, or both of infected pigs relative to controls occurred for all but one gene. Expression differences were greater in HD than I pigs. Significant downregulation for certain immune genes in low pigs, relative to littermate controls, was detected in lung and BLN, particularly in line I. Serum levels of the immune cytokines affirmed the gene expression differences. High preinfection serum levels of IL 8 were significantly associated with PRRS virus-resistant, low pigs. After infection, low expression of interferon gamma in cDNA and in serum was also correlated with PRRS virus resistance. Important genetic associations were revealed for fine mapping of candidate genes for PRRS virus resistance and determining the causative alleles.