A retrospective study suggests 55 days of persistence of SARS-CoV-2 during the first wave of the pandemic in Santiago de Chile.

Background: As the COVID-19 pandemic persists, infections continue to surge globally. Presently, the most effective strategies to curb the disease and prevent outbreaks involve fostering immunity, promptly identifying positive cases, and ensuring their timely isolation. Notably, there are instances where the SARS-CoV-2 virus remains infectious even after patients have completed their quarantine. Objective: Understanding viral persistence post-quarantine is crucial as it could account for localized infection outbreaks. Therefore, studying and documenting such instances is vital for shaping future public health policies. Design: This study delves into a unique case of SARS-CoV-2 persistence in a 60-year-old female healthcare worker with a medical history of hypertension and hypothyroidism. The research spans 55 days, marking the duration between her initial and subsequent diagnosis during Chile's first COVID-19 wave, with the analysis conducted using RT-qPCR. Results: Genomic sequencing-based phylogenetic analysis revealed that the SARS-CoV-2 detected in both Nasopharyngeal swab samples (NPSs) was consistent with the 20B clade of the Nextstrain classification, even after a 55-day interval. Conclusion: This research underscores the need for heightened vigilance concerning cases of viral persistence. Such instances, albeit rare, might be pivotal in understanding sporadic infection outbreaks that occur post-quarantine.

An ecological study on reinfection rates using a large dataset of RT-qPCR tests for SARS-CoV-2 in Santiago of Chile.

Introduction: As the SARS-CoV-2 continues to evolve, new variants pose a significant threat by potentially overriding the immunity conferred by vaccination and natural infection. This scenario can lead to an upswing in reinfections, amplified baseline epidemic activity, and localized outbreaks. In various global regions, estimates of breakthrough cases associated with the currently circulating viral variants, such as Omicron, have been reported. Nonetheless, specific data on the reinfection rate in Chile still needs to be included. Methods: Our study has focused on estimating COVID-19 reinfections per wave based on a sample of 578,670 RT-qPCR tests conducted at the University of Santiago of Chile (USACH) from April 2020 to July 2022, encompassing 345,997 individuals. Results: The analysis reveals that the highest rate of reinfections transpired during the fourth and fifth COVID-19 waves, primarily driven by the Omicron variant. These findings hold despite 80% of the Chilean population receiving complete vaccination under the primary scheme and 60% receiving at least one booster dose. On average, the interval between initial infection and reinfection was found to be 372 days. Interestingly, reinfection incidence was higher in women aged between 30 and 55. Additionally, the viral load during the second infection episode was lower, likely attributed to Chile's high vaccination rate. Discussion: This study demonstrates that the Omicron variant is behind Chile's highest number of reinfection cases, underscoring its potential for immune evasion. This vital epidemiological information contributes to developing and implementing effective public health policies.

Genomic Evidence Suggests Viral Persistence of SARS-CoV-2 for 386 Days in Health Worker: A Case Report from Santiago of Chile.

The COVID-19 pandemic continues to affect several countries. One of the best ways to control its spread is the timely identification of infected patients for isolation and quarantine. While an episode of infection lasts an average of 8-10 days from the onset of symptoms, there is literature describing long-lasting viral persistence events. Here, we report a case of persistence of SARS-CoV-2 for 386 days in a health worker from Santiago de Chile. Our study could be one of the longest reported viral persistence events. RNA sequencing analyses indicated that the first positive diagnosis (8 June 2020) corresponded to a SARS-CoV-2 variant belonging to Clade Nextstrain 20A. Three hundred eighty-six days later (23 September 2021), the second positive result reached the same viral variant (Clade 20A) but without presence or circulation in Chile since May 2021. Both sequencing coverages showed an identity of 99.21%, with some mutations related to the severity of the disease (ORF1b:P314L) and more infectivity (S:D614G). This work reinforces the idea of implementing an RT-qPCR or rapid antigen test once the quarantine is fulfilled to ensure viral absence, identify potential persistence, and, consequently, minimize the risk of local outbreaks of SARS-CoV-2 infection.

Evaluation and comparison of the sensitivity of three commercial RT-qPCR kits used for the detection of SARS-CoV-2 in Santiago, Chile.

Introduction: The COVID-19 pandemic is still in force, causing global public health challenges and threats. Although vaccination and herd immunity have proven to be the most efficient way to control the pandemic, massive and early testing of patients using the RT-qPCR technique is crucial for constant genomic surveillance. The appearance of variants of SARS-CoV-2 with new mutations can reduce the efficiency of diagnostic detection. In this sense, several commercial RT-qPCR kits have been the target of extensive analysis because low assay performance could lead to false-negative diagnoses. Methods: In this study, we evaluated the performance of three commercial RT-qPCR kits; Thermo Fisher (TaqMan 2019-nCoV Assay Kit v1), BGI and Roche (LightCycler® Multiplex RNA Virus Master) used for the diagnosis of COVID-19 throughout the pandemic in Santiago de Chile. Results: Under our best assay conditions, we found significant differences in Cq amplification values for control and viral probes, against the same nasopharyngeal swab samples (NPSs). In addition, in some cases, the sensitivity of the RT-qPCR kits decreased against viral variants. Conclusion: Our study suggests evaluating the RT-qPCR kits used to detect SARS-CoV-2 because variants such as Omicron, which has several mutations, can compromise their detection and underestimate viral circulation.

Infectious pancreatic necrosis virus (IPNV) recombinant viral protein 1 (VP1) and VP2-Flagellin fusion protein elicit distinct expression profiles of cytokines involved in type 1, type 2, and regulatory T cell response in rainbow trout (Oncorhynchus mykiss).

In this study, we examined the cytokine immune response against two proteins of infectious pancreatic necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss), the virion-associated RNA polymerase VP1 and VP2-Flagellin (VP2-Flg) fusion protein. Since VP1 is not a structural protein, we hypothesize it can induce cellular immunity, an essential mechanism of the antiviral response. At the same time, the fusion construction VP2-Flg could be highly immunogenic due to the presence of the flagellin used as an adjuvant. Fish were immunized with the corresponding antigen in Montanide™, and the gene expression of a set of marker genes of Th1, Th2, and the immune regulatory response was quantified in the head kidney of immunized and control fish. Results indicate that VP1 induced upregulation of ifn-γ, il-12p40c, il-4/13a, il-4/13b2, il-10a, and tgf-ß1 in immunized fish. Expression of il-2a did not change in treated fish at the times tested. The antigen-dependent response was analysed by in vitro restimulation of head kidney leukocytes. In this assay, the group of cytokines upregulated after VP1-restimulation was consistent with those upregulated in the head kidney in vivo. Interestingly, VP1 induced il-2a expression after in vitro restimulation. The analysis of sorted lymphocytes showed that the increase of cytokines occurred in CD4-1+ T cells suggesting that Th differentiation happens in response to VP1. This is also consistent with the expression of t-bet and gata3, the master regulators for Th1/Th2 differentiation in the kidneys of immunized animals. A different cytokine expression profile was found after VP2-Flg administration, i.e., upregulation occurs for ifn-γ, il-4/13a, il-10a, and tgf-ß1, while down-regulation was observed in il-4/13b2 and il-2a. The cytokine response was due to flagellin; only the il-2a effect was dependent upon VP2 in the fusion protein. To the best of our knowledge this study reports for the first-time characteristics of the adaptive immune response induced in response to IPNV VP1 and the fusion protein VP2-Flg in fish. VP1 induces cytokines able to trigger the humoral and cell-mediated immune response in rainbow trout. The analysis of the fish response against VP2-Flg revealed the immunogenic properties of Aeromonas salmonicida flagellin, which can be further tested for adjuvanticity. The novel immunogenic effects of VP1 in rainbow trout open new opportunities for further IPNV vaccine development using this viral protein.

Sensitivity analysis of rapid antigen tests for the Omicron SARS-CoV-2 variant detection from nasopharyngeal swab samples collected in Santiago of Chile.

The COVID-19 pandemic continues to be a concern and keeps global health authorities on alert. The RT-PCR technique has been the gold-standard assay for detecting the SARS-CoV-2 virus. However, rapid antigen tests (RATs) have been widely used to increase the number of tests faster and more efficiently in the population. Nevertheless, the appearance of new viral variants, with genomic mutations associated with greater contagiousness and immune evasion, highlights the need to evaluate the sensitivity of these RATs. This report evaluates the sensitivity of SD Biosensor-Roche, Panbio™, and Clinitest® RATs widely used in Santiago de Chile in the detection of the Omicron variant from Nasopharyngeal samples (NPSs), the most predominant SARS-CoV-2 variant in Chile and the world. SD Biosensor-Roche shows a detection sensitivity of 95.7% in the viral amplification range of 20 ≤ Cq < 25, while Panbio™ and Clinitest® show 100% and 91.3%, respectively. In the viral amplification ranges of 25 ≤ Cq < 30, the detection sensitivity decreased to 28% for SD Biosensor-Roche, 32% for Panbio™, and 72% for Clinitest®. This study indicates that the tested RATs have high sensitivity in detecting the Omicron variant of concern (VOC) at high viral loads. By contrast, its sensitivity decreases at low viral loads. Therefore, it is suggested to limit the use of RATs as an active search method, considering that infections in patients are increasingly associated with lower viral loads of SARS-CoV-2. These antecedents could prevent contagion outbreaks and reduce the underestimation of the current Omicron variant circulation at the local level.

Epidemiological characteristics of Omicron and Delta SARS-CoV-2 variant infection in Santiago, Chile.

The variant of concern (VOC) SARS-CoV-2 Omicron (B.1.1529) has been described as a highly contagious variant but less virulent than the current variant being monitored (VBM) Delta (B.1.617.2), causing fewer cases of hospitalizations, symptomatology, and deaths associated with COVID-19 disease. Although the epidemiological comparison of both variants has been previously reported in other countries, no report indicates their behavior and severity of infection in Chile. In this work, we report for the first time the effect of the Omicron and Delta variants in a cohort of 588 patients from the Hospital de Urgencia Asistencia pública (HUAP), a high-complexity health center in Santiago, Chile. This report is framed at the beginning of Chile's third wave of the COVID-19 pandemic, with a marked increase in the Omicron variant and a decrease in the circulating Delta variant. Our results indicated a similar proportion of patients with a complete vaccination schedule for both variants. However, the Delta variant was associated with a higher prevalence of hospitalization and more significant symptomatology associated with respiratory distress. On the other hand, our data suggest that vaccination is less effective in preventing infection by the Omicron variant. This antecedent, with a low severity but high contagiousness, suggests that the Omicron variant could even collapse the primary health care service due to the high demand for health care.

The Comparative Analysis of Two RT-qPCR Kits for Detecting SARS-CoV-2 Reveals a Higher Risk of False-Negative Diagnosis in Samples with High Quantification Cycles for Viral and Internal Genes.

The early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using the real-time quantitative polymerase chain reaction (RT-qPCR) as a gold-standard molecular tool has allowed to test and trace the viral spread and the isolation of COVID-19-infected patients. The detection capacity of viral and internal genes is an essential parameter to consider and analyze during the assay. In this study, we analyze the performance of the two commercial RT-qPCR kits used in Chile, TaqMan™ 2019-nCoV Control Kit v1 (Thermo Fisher) and MaxCov19 (TAAG Genetics), for the COVID-19 diagnosis from nasopharyngeal swab samples (NPSs). Our results show a lower sensitivity of the TAAG kit compared to the Thermo Fisher kit, even in the detection of SARS-CoV-2 mutations associated with its variants. This study reinforces the relevance of evaluating the performance of RT-qPCR kits before being used massively since those with lower sensitivity can generate false negatives and produce outbreaks of local infections.

Comparison of the First and Second Wave of Infections by SARS-CoV-2: A Retrospective and Longitudinal Study From a Primary Health Care Center in Santiago of Chile.

The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Many countries have reported the experience of at least two contagion waves, describing associated mortality rates and population behavior. The analysis of the effect of this pandemic in different localities can provide valuable information on the key factors to consider in the face of future massive infectious diseases. This work describes the first retrospective and comparative study about behavior during the first and second waves of the COVID-19 pandemic in Chile from a primary Healthcare Center. From 19,313 real-time quantitative PCR (RT-qPCR) tests assessed, the selected 1,694 positive diagnostics showed a decrease in mortality rate in the second wave (0.6%) compared with the first (4.6%). In addition, we observed that infections in the second wave were mainly in young patients with reduced comorbidities. The population with a complete vaccination schedule shows a decrease in the duration of symptoms related to the disease, and patients with more comorbidities tend to develop severe illness. This report provides evidence to partially understand the behavior and critical factors in the severity of the COVID-19 pandemic in the population of Santiago of Chile.

First Identification of Reinfection by a Genetically Different Variant of SARS-CoV-2 in a Homeless Person from the Metropolitan Area of Santiago, Chile.

The identification and tracking of SARS-CoV-2 infected patients in the general population are essential components of the global strategy to limit the COVID-19 viral spread, specifically for maintaining traceability and suppressing the resurgence of local outbreaks. Public health programs that include continuous RT-qPCR testing for COVID-19 in the general population, viral sequencing, and genomic surveillance for highly contagious forms of the virus have allowed for the identification of SARS-CoV-2 infections and reinfections. This work identified SARS-CoV-2 reinfection in a homeless person, which occurred 58 days after the first COVID-19 diagnosis. Genomic sequencing identified a different Nextstrain classification clade (20A and 20B) and PANGO lineage, with a divergence of 4 single nucleotide variants (SNVs) in S and ORF1ab genes, suggesting reinfection by different viral variants. This study is the first from the great metropolitan area of Santiago, Chile, one of the top ten countries in the world to live during the COVID-19 pandemic. We support the importance of performing intensive genomic surveillance programs in the whole population and high-risk groups, such as homeless people, nearly 20 thousand people in Chile, and have limited access to health care services and poor viral traceability.

Description of Symptoms Caused by the Infection of the SARS-CoV-2 B.1.621 (Mu) Variant in Patients With Complete CoronaVac Vaccination Scheme: First Case Report From Santiago of Chile.

Vaccine administration is one of the most efficient ways to control the current coronavirus disease 2019 (COVID-19) pandemic. However, the appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants can avoid the immunity generated by vaccines. Thus, in patients with a complete vaccine schedule, the infection by SARS-CoV-2 may cause severe, mild, and asymptomatic manifestations of the disease. In this case report, we describe for the first time the clinical symptoms of four patients (three symptomatic; one asymptomatic) from Santiago of Chile, with a complete vaccination schedule with two doses of CoronaVac (Sinovac Life Science) infected with the variant of interest (VOI) B.1.621 (Mu). They were compared with four unvaccinated patients, who had a higher prevalence of symptoms after infection compared to vaccinated patients. In the CoronaVac-vaccinated group, an 80-year-old patient who registered various comorbidities required Invasive mechanical ventilation for 28 days with current home medical recovery discharge. By contrast, in the unvaccinated group, a 71-year-old presented more symptoms with more than 45 days of Invasive mechanical ventilation, which continues to date, presenting greater lung damage than the vaccinated hospitalized patient. This first report evidence differences in the clinical symptomatology of patients vaccinated and non-vaccinated infected with the VOI B.1.621 (Mu) and suggest the protective effects of CoronaVac against this variant.

Sleeping With the Enemy? The Current Knowledge of Piscine Orthoreovirus (PRV) Immune Response Elicited to Counteract Infection.

Piscine orthoreovirus (PRV) is a virus in the genus Orthoreovirus of the Reoviridae family, first described in 2010 associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic salmon (Salmo salar). Three phases of PRV infection have been described, the early entry and dissemination, the acute dissemination phase, and the persistence phase. Depending on the PRV genotype and the host, infection can last for life. Mechanisms of immune response to PRV infection have been just beginning to be studied and the knowledge in this matter is here revised. PRV induces a classical antiviral immune response in experimental infection of salmonid erythrocytes, including transcriptional upregulation of ifn-α, rig-i, mx, and pkr. In addition, transcript upregulation of tcra, tcrb, cd2, il-2, cd4-1, ifn-γ, il-12, and il-18 has been observed in Atlantic salmon infected with PRV, indicating that PRV elicited a Th1 type response probably as a host defense strategy. The high expression levels of cd8a, cd8b, and granzyme-A in PRV-infected fish suggest a positive modulatory effect on the CTL-mediated immune response. This is consistent with PRV-dependent upregulation of the genes involved in antigen presentation, including MHC class I, transporters, and proteasome components. We also review the potential immune mechanisms associated with the persistence phenotype of PRV-infected fish and its consequence for the development of a secondary infection. In this scenario, the application of a vaccination strategy is an urgent and challenging task due to the emergence of this viral infection that threatens salmon farming.

The RNA-dependent RNA polymerase of the infectious pancreatic necrosis virus is linked to viral mRNA acting as a cap substitute.

The infectious pancreatic necrosis virus (IPNV) is responsible for significant economic losses in the aquaculture industry. It is an unenveloped virus with an icosahedral capsid. Its viral genome comprises two dsRNA segments, A and B. Segment A contains a small ORF, which encodes VP5, and a large ORF, which encodes a polyprotein that generates the structural proteins and the viral protease. Segment B encodes the RNA-dependent RNA polymerase (RdRp), called VP1 in this free form, or Vpg when it covalently attaches to the viral RNA. The viral genome does not have cap or poly(A). Instead, each 5' end is linked to the Vpg. Recently, we demonstrated that mRNA-A contains an internal ribosome entry site (IRES) to command polyprotein synthesis. However, the presence of Vpg on IPNV mRNAs and its impact on cellular translation has not been investigated. This research demonstrates that IPNV mRNAs are linked to Vpg and that this protein inhibits cap-dependent translation on infected cells. Also, it is demonstrated that Vpg interacts with eIF4E and that rapamycin treatment partially diminishes the viral protein synthesis. In addition, we determined that an IRES does not command translation of IPNV mRNA-B. We show that VPg serves as a cap substitute during the initiation of IPNV translation, contributing to understanding the replicative cycle of Birnaviruses. Our results indicate that the viral protein VP1/Vpg is multifunctional, having a significant role during IPNV RNA synthesis as the RdRp and the primer for IPNV RNA synthesis and translation as the viral protein genome, acting as a cap substitute.

Lactococcus lactis Expressing Type I Interferon From Atlantic Salmon Enhances the Innate Antiviral Immune Response In Vivo and In Vitro.

In salmon farming, viruses are responsible for outbreaks that produce significant economic losses for which there is a lack of control tools other than vaccines. Type I interferon has been successfully used for treating some chronic viral infections in humans. However, its application in salmonids depends on the proper design of a vehicle that allows its massive administration, ideally orally. In mammals, administration of recombinant probiotics capable of expressing cytokines has shown local and systemic therapeutic effects. In this work, we evaluate the use of Lactococcus lactis as a type I Interferon expression system in Atlantic salmon, and we analyze its ability to stimulate the antiviral immune response against IPNV, in vivo and in vitro. The interferon expressed in L. lactis, even though it was located mainly in the bacterial cytoplasm, was functional, stimulating Mx and PKR expression in CHSE-214 cells, and reducing the IPNV viral load in SHK-1 cells. In vivo, the oral administration of this L. lactis producer of Interferon I increases Mx and PKR expression, mainly in the spleen, and to a lesser extent, in the head kidney. The oral administration of this strain also reduces the IPNV viral load in Atlantic salmon specimens challenged with this pathogen. Our results show that oral administration of L. lactis producing Interferon I induces systemic effects in Atlantic salmon, allowing to stimulate the antiviral immune response. This probiotic could have effects against a wide variety of viruses that infect Atlantic salmon and also be effective in other salmonids due to the high identity among their type I interferons.

The Analysis of Live-Attenuated Piscirickettsia salmonis Vaccine Reveals the Short-Term Upregulation of Innate and Adaptive Immune Genes in Atlantic Salmon (Salmo salar): An In Situ Open-Sea Cages Study.

Piscirickettsia salmonis, the etiological agent of the Salmon Rickettsial Septicemia (SRS), is one the most serious health problems for the Chilean salmon industry. Typical antimicrobial strategies used against P. salmonis include antibiotics and vaccines, but these applications have largely failed. A few years ago, the first attenuated-live vaccine against SRS (ALPHA JECT LiVac® SRS vaccine) was released to the market. However, there is no data about the agents involved in the activation of the immune response induced under field conditions. Therefore, in this study we evaluated the expression profile of a set of gene markers related to innate and adaptive immunity in the context of a cellular response in Atlantic salmon (Salmo salar) reared under productive farm conditions and immunized with a live-attenuated vaccine against P. salmonis. We analyzed the expression at zero, 5-, 15- and 45-days post-vaccination (dpv). Our results reveal that the administration of the attenuated live SRS LiVac vaccine induces a short-term upregulation of the cellular-mediated immune response at 5 dpv modulated by the upregulation of ifnα, ifnγ, and the cd4 and cd8α T cell surface markers. In addition, we also registered the upregulation of il-10 and tgfß. Altogether, the results suggest that a balanced activation of the immune response took place only at early times post-vaccination (5 dpv). The scope of this short-term upregulation of the cellular-mediated immune response against a natural outbreak in fish subjected to productive farm conditions deserves further research.

The Rapid Antigen Detection Test for SARS-CoV-2 Underestimates the Identification of COVID-19 Positive Cases and Compromises the Diagnosis of the SARS-CoV-2 (K417N/T, E484K, and N501Y) Variants.

Timely detection of severe acute respiratory syndrome due to coronavirus 2 (SARS-CoV-2) by reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been the gold- strategy for identifying positive cases during the current pandemic. However, faster and less expensive methodologies are also applied for the massive diagnosis of COVID-19. In this way, the rapid antigen test (RAT) is widely used. However, it is necessary to evaluate its detection efficiency considering the current pandemic context with the circulation of new viral variants. In this study, we evaluated the sensitivity and specificity of RAT (SD BIOSENSOR, South Korea), widely used for testing and SARS-CoV-2 diagnosis in Santiago of Chile. The RAT showed a 90% (amplification range of 20 ≤ Cq <25) and 10% (amplification range of 25 ≤ Cq <30) of positive SARS-CoV-2 cases identified previously by RT-qPCR. Importantly, a 0% detection was obtained for samples within a Cq value>30. In SARS-CoV-2 variant detection, RAT had a 42.8% detection sensitivity in samples with RT-qPCR amplification range 20 ≤ Cq <25 containing the single nucleotide polymorphisms (SNP) K417N/T, N501Y and E484K, associated with beta or gamma SARS-CoV-2 variants. This study alerts for the special attention that must be paid for the use of RAT at a massive diagnosis level, especially in the current scenario of appearance of several new SARS-CoV-2 variants which could generate false negatives and the compromise of possible viral outbreaks.

The Atlantic salmon interleukin 4/13 receptor family: Structure, tissue distribution and modulation of gene expression.

Interleukin (IL)-4 and IL-13 play a central role in T helper 2 immune response in mammals. The cell signalling is mediated by the type I heterodimeric receptor containing the IL-4Rα and γC chains, and the type II receptors formed by IL-4Rα and IL-13Rα1. In salmonid species, three paralogues of the IL-4 and IL-13 cytokines have been reported, il-4/13a, il-4/13b1 and il-4/13b2. In regard to receptors, two paralogues of each IL-4/13 receptor chains have been identified in rainbow trout while five genes named γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 have identified in Atlantic salmon. Since Atlantic salmon is an important farmed fish species, the aim of this work was to get new insights into distribution, structure and expression regulation of the IL-4/13 receptors in salmon. By using qRT-PCR, it was shown that all γc1, il-4rα, il-13rα1a, il-13rα1b, and il-13rα2 receptor chains were expressed in lymphoid and non-lymphoid tissues of healthy salmon, nonetheless γC expression was higher in lymphoid than non-lymphoid tissues. The in silico structural analysis and homology modelling of the predicted receptor proteins showed that domains and most motifs present in the superior vertebrate chains are conserved in salmon suggesting a conserved role for these receptor chains. Only IL-13Rα1B is a receptor chain with a unique structure that seem not to be present in higher vertebrates but in fish species. In order to determine the regulatory role of IL-4/13 on the expression of receptor chains, Atlantic salmon il-4/13A gene was synthetized and cloned in pET15b. The recombinant IL-4/13A was produced in E. coli and the activity of the purified cytokine was confirmed in vitro. The regulatory role of IL-4/13A on the expression of their potential receptors was tested in salmon receiving the recombinant cytokine and effects were compared with those of the control group. The results showed that IL-4/13A induced the expression of its own gene and GATA-3, in the head kidney of fish but not in the spleen, while IL-10 increased in both lymphoid organs indicating a regulatory role of this cytokine on the induction of Th2 responses in salmon. IFN-γ and MHC class II were also later induced in head kidney. In regard to the expression of the receptor chains, IL-4/13A upregulated the expression of γC, IL-13Rα1A and IL-13Rα2A in the spleen but not in the head kidney of salmon, indicating a role on the modulation of cell signalling for the Th2 response. Furthermore, Piscirickettsia salmonis infection of Atlantic salmon occurred with an increase of γC and IL-13Rα1A suggesting a potential role of the IL-4/13 system in bacterial immunity or pathogenesis. This study contributes to a better understanding of the IL-4/13A system in salmon, which as a key axis for Th2 response may be involved not only in pathogen elimination but also in adaptive immune repair that seems critical tolerance to infectious diseases.

Effect of yeast (Xanthophyllomyces dendrorhous) and plant (Saint John's wort, lemon balm, and rosemary) extract based functional diets on antioxidant and immune status of Atlantic salmon (Salmo salar) subjected to crowding stress.

Salmon farming may face stress due to the intensive culture conditions with negative impacts on overall performance. In this aspect, functional feed improves not only the basic nutritional requirements but also the health status and fish growth. However, to date no studies have been carried out to evaluate the effect of functional diets in salmon subjected to crowding stress. Thus, the aim of this study was to evaluate the effect of yeast extract (Xanthophyllomyces dendrorhous; diet A) and the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary; diet B) on the antioxidant and immune status of Atlantic salmon grown under normal cultured conditions and then subjected to crowding stress. Fish were fed with functional diets during 30 days (12 kg/m3) and then subjected to crowding stress (20 kg/m3) for 10 days. The lipid peroxidation in gut showed that both diets induced a marked decrease on oxidative damage when fish were subjected to crowding stress. The protein carbonylation in muscle displayed at day 30 a marked decrease in both functional diets that was more marked on the stress condition. The expression of immune markers (IFNγ, CD4, IL-10, TGF-ß, IgMmb, IgMsec, T-Bet, and GATA-3) indicated the upregulation of those associated to humoral-like response (CD4, IL-10, GATA-3) when fish were subjected to crowding stress. These results were confirmed with the expression of secreted IgM. Altogether, these functional diets improved the antioxidant status and increased the expression of genes related to Th2-like response suggesting a protective role on fish subjected to crowding stress.

Study of RNA-A Initiation Translation of The Infectious Pancreatic Necrosis Virus.

The infectious pancreatic necrosis virus (IPNV) is a salmonid pathogen that causes significant economic losses to the aquaculture industry. IPNV is a non-enveloped virus containing two uncapped and non-polyadenylated double strand RNA genomic segments, RNA-A and RNA-B. The viral protein Vpg is covalently attached to the 5' end of both segments. There is little knowledge about its viral cycle, particularly about the translation of the RNAs. Through experiments using mono and bicistronic reporters, in this work we show that the 120-nucleotide-long 5'-UTR of RNA-A contains an internal ribosome entry site (IRES) that functions efficiently both in vitro and in salmon cells. IRES activity is strongly dependent on temperature. Also, the IRES structure is confined to the 5'UTR and is not affected by the viral coding sequence. This is the first report of IRES activity in a fish virus and can give us tools to generate antivirals to attack the virus without affecting fish directly.

ß-1,3/1,6-Glucan-supplemented diets antagonize immune inhibitory effects of hypoxia and enhance the immune response to a model vaccine.

The diets of farmed salmon are usually supplemented with immunostimulants to improve health status. Because ß-glucan is one of the most common immunostimulants used in diets, here we examined the effect of two ß-1,3/1,6-glucan-supplemented diets on the expression of immune response genes of Atlantic salmon. The relative abundances of IFN-α1, Mx, IFN-γ, IL-12, TGF-ß1, IL-10, and CD4 transcripts were evaluated in head kidney by qRT-PCR. We assessed the effects of the diets under normoxia and acute hypoxia, as salmon are especially sensitive to changes in the concentration of dissolved oxygen, which can also affect immunity. These effects were also tested on vaccinated fish, as we expected that ß-1,3/1,6-glucan-supplemented diets would enhance the adaptive immune response to the vaccine. We found that administration of the Bg diet (containing ß-1,3/1,6-glucan) under normoxia had no effects on the expression of the analyzed genes in the kidney of the diet-fed fish, but under hypoxia/re-oxygenation (90 min of acute hypoxia), the ßg diet affected the expression of the antiviral genes, IFN-α1 and Mx, preventing their decrease caused by hypoxia. The Bax diet, which in addition to ß-1,3/1,6-glucan, contained astaxanthin, increased IL-12 and IFN-γ in kidneys. With fish exposed to hypoxia/reoxygenation, the diet prevented the decrease of IFN-α1 and Mx levels observed after hypoxia. When fish were vaccinated, only the levels of IL-12 and CD4 transcripts increased, but interestingly if fish were also fed the Bax diet, the vaccination induced a significant increase in all the analyzed transcripts. Finally, when vaccinated fish were exposed to hypoxia, the effect of the Bax diet was greatly reduced for all genes tested and moreover, inducible effects completely disappeared for IL-12, IFN-α, and Mx. Altogether, these results showed that the tested ß-1,3/1,6-glucan diets increased the levels of transcripts of key genes involved in innate and adaptive immune response of salmon, potentiating the response to a model vaccine and also antagonizing the effects of hypoxia.