Water-in-oil adjuvant challenges in fish vaccination: An experimental inactivated adjuvanted vaccine against betanodavirus infection in Senegalese sole.

The extensive growth of intensive fish farming has led to a massive spread of infectious diseases. Nervous necrosis virus (NNV) is the causative agent of the viral encephalo- and retinopathy disease which has become a major threat for fish farming all over the globe. The devastating mortality rates recorded in disease outbreaks, especially when infected specimens are at early stages of development, have a high economic impact on the sector. Currently, vaccines are the most cost-effective preventing tool in the fight against viruses. Inactivated vaccines have the advantage of simplicity in their development at the same time as present the antigen in a similar manner than the natural infection in the host. Nevertheless, they usually trigger weaker immune responses needing adjuvants to boost their effectiveness. In this work, we have intraperitoneally vaccinated Senegalese sole juveniles (Solea senegalensis) with a previously designed inactivated vaccine against NNV based on binary ethylenimine (BEI), mixed or not with an oil-adjuvant. Our results demonstrated the potential activation of different immune pathways when the vaccine was administered alone compared to the oil-adjuvanted vaccine, both resulting in an equivalent partial improvement in survival following a NNV challenge. However, whilst the vaccine alone led to a significant increase in specific antibodies, in the adjuvanted version those antibodies were kept basal although with a slight improvement in their neutralization capacity. At transcriptional level, neither vaccine (adjuvanted or not) triggered the immune system activation during the vaccination period. However, after NNV infection, the BEI-inactivated vaccines alone and oil-adjuvanted both elicited the stimulation of antiviral responsive genes (rtp3, herc4), antigen presentation molecules (mhcii) and T-cell markers (cd8a) in the head-kidney. Additionally, the oil-adjuvanted vaccine appears to stimulate mediator cytokines (il6) and B-cell markers (ight and ighm). Surprisingly, when the adjuvant was administered alone, fish showed the highest survival rates concomitantly with a lack of NNV-IgM production, pointing to the possible induction of different immune pathways than the B-cell responses via antibodies by the adjuvant. Since this combined vaccine did not succeed in the full extension of protection against the pathogen, further studies should be performed focusing on unravelling the molecular mechanisms through which adjuvants trigger the immune response, both independently and when added to a vaccine antigen.

Antiviral Activity of Carrageenans and Processing Implications.

Carrageenan and carrageenan oligosaccharides are red seaweed sulfated carbohydrates with well-known antiviral properties, mainly through the blocking of the viral attachment stage. They also exhibit other interesting biological properties and can be used to prepare different drug delivery systems for controlled administration. The most active forms are λ-, ι-, and κ-carrageenans, the degree and sulfation position being determined in their properties. They can be obtained from sustainable worldwide available resources and the influence of manufacturing on composition, structure, and antiviral properties should be considered. This review presents a survey of the antiviral properties of carrageenan in relation to the processing conditions, particularly those assisted by intensification technologies during the extraction stage, and discusses the possibility of further chemical modifications.

Psychometric properties of the European Portuguese version of the Wijma Delivery Expectancy/Experience Questionnaire in pregnant women.

The researchers' aim is to examine the psychometric properties of the European Portuguese version of the Wijma Delivery Expectancy/Experience Questionnaire version A (WDEQ-A). Data from 669 pregnant Portuguese women was collected between June and October of 2019. From the initial 33-item Portuguese version of the WDEQ-A, a 27-item instrument was developed. Five-factor solution explained 63.8% of the total variance. The factors defined by the researchers were: fear/lack of self-efficacy, loneliness, negative appraisal, lack of positive anticipation and concern for the child. Cronbach's alpha coefficient was 0.934. The European Portuguese version of the WDEQ-A is a reliable and valid tool to measure fear of childbirth on both nulliparous and multiparous women and can be a useful tool for cross-cultural research. Moreover, researchers support the use of this tool in clinical practice to recognize and address fearful women during prenatal period.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

Amino acidic substitutions in the polymerase N-terminal region of a reassortant betanodavirus strain causing poor adaptation to temperature increase.

Nervous necrosis virus (NNV), Genus Betanodavirus, is the causative agent of viral encephalopathy and retinopathy (VER), a neuropathological disease that causes fish mortalities worldwide. The NNV genome is composed of two single-stranded RNA molecules, RNA1 and RNA2, encoding the RNA polymerase and the coat protein, respectively. Betanodaviruses are classified into four genotypes: red-spotted grouper nervous necrosis virus (RGNNV), striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV) and tiger puffer nervous necrosis virus (TPNNV). In Southern Europe the presence of RGNNV, SJNNV and their natural reassortants (in both RNA1/RNA2 forms: RGNNV/SJNNV and SJNNV/RGNNV) has been reported. Pathology caused by these genotypes is closely linked to water temperature and the RNA1 segment encoding amino acids 1-445 has been postulated to regulate viral adaptation to temperature. Reassortants isolated from sole (RGNNV/SJNNV) show 6 substitutions in this region when compared with the RGNNV genotype (positions 41, 48, 218, 223, 238 and 289). We have demonstrated that change of these positions to those present in the RGNNV genotype cause low and delayed replication in vitro when compared with that of the wild type strain at 25 and 30 °C. The experimental infections confirmed the impact of the mutations on viral replication because at 25 °C the viral load and the mortality were significantly lower in fish infected with the mutant than in those challenged with the non-mutated virus. It was not possible to challenge fish at 30 °C because of the scarce tolerance of sole to this temperature.

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo.

Betanodavirus reassortant strains (RGNNV/SJNNV) isolated from Senegalese sole harbour an SJNNV capsid featuring several changes with respect to the SJNNV-type strain, sharing three hallmark substitutions. Here, we have employed recombinant strains harbouring mutations in these positions (r20 and r20 + 247 + 270) and have demonstrated that the three substitutions affect different steps of the viral replication process. Adsorption ability and efficiency of viral attachment were only affected by substitutions in the C-terminal side of the capsid. However, the concurrent mutation in the N-terminal side seems to slightly decrease these properties, suggesting that this region could also be involved in viral binding. Differences in the intracellular and extracellular production of the mutant strains suggest that both the C-terminal and N-terminal regions of the capsid protein may be involved in the particle budding. Furthermore, viral replication in sole brain tissue of the mutant strains, and especially double- and triple-mutant strains, is clearly delayed with respect to the wt strain. These data support previous findings indicating that the C-terminal side plays a role in virulence because of a slower spread in the fish host brain and suggest that the concurrent participation of the N-terminal side is also important for viral replication in vivo.

Modification of betanodavirus virulence by substitutions in the 3' terminal region of RNA2.

Betanodaviruses have bi-segmented positive-sense RNA genomes, consisting of RNAs 1 and 2. For some members of the related genus alphanodavirus, the 3' terminal 50 nucleotides (nt) of RNA2, including a predicted stem-loop structure (3'SL), are essential for replication. We investigate the possible existence and role of a similar structure in a reassortant betanodavirus strain (RGNNV/SJNNV). In this study, we developed three recombinant strains containing nucleotide changes at positions 1408 and 1412. Predictive models showed stem-loop structures involving nt 1398-1421 of the natural reassortant whereas this structure is modified in the recombinant viruses harbouring point mutations r1408 and r1408-1412, but not in r1412. Results obtained from infectivity assays showed differences between the reference strains and the mutants in both RNA1 and RNA2 synthesis. Moreover, an imbalance between the synthesis of both segments was demonstrated, mainly with the double mutant. All these results suggest an interaction between RNA1 and the 3' non-coding regions (3'NCR) of RNA2. In addition, the significant attenuation of the virulence for Senegalese sole and the delayed replication of r1408-1412 in brain tissues may point to an interaction of RNA2 with host cellular proteins.

Betanodavirus infection in primary neuron cultures from sole.

Nervous necrosis virus (NNV), G. Betanodavirus, is the causative agent of viral encephalopathy and retinopathy, a disease that causes mass mortalities in a wide range of fish species. Betanodaviruses are neurotropic viruses and their replication in the susceptible fish species seems to be almost entirely restricted to nerve tissue. However, none of the cell lines used for NNV propagation has a nervous origin. In this study, first we established a protocol for the primary culture of neurons from Senegalese sole, which made it possible to further study virus-host cell interactions. Then, we compared the replication of three NNV strains with different genotypes (SJNNV, RGNNV and a RGNNV/SJNNV reassortant strain) in sole neuron primary cultures and E-11 cells. In addition, to study how two amino acid substitutions at the c-terminal of the capsid protein (positions 247 and 270) affect the binding to cell receptors, a recombinant strain was also tested. The results show that sole neural cells enabled replication of all the tested NNV strains. However, the recombinant strain shows a clearly delayed replication when compared with the wt strain. This delay was not observed in virus replicating in E-11 cells, suggesting a viral interaction with different cell receptors. The establishment of a sole primary neuronal culture protocol provides an important tool for research into betanodavirus infection in sole.

Betanodavirus infection in bath-challenged Solea senegalensis juveniles: A comparative analysis of RGNNV, SJNNV and reassortant strains.

Senegalese sole has been shown to be highly susceptible to betanodavirus infection, although virulence differences were observed between strains. To study the mechanisms involved in these differences, we have analysed the replication in brain tissue of three strains with different genotypes during 15 days after bath infection. In addition, possible portals of entry for betanodavirus into sole were investigated. The reassortant RGNNV/SJNNV and the SJNNV strain reached the brain after 1 and 2 days postinfection, respectively. Although no RGNNV replication was detected until day 3-4 postinfection, at the end of the experiment this strain yielded the highest viral load; this is in accordance with previous studies in which sole infected with the reassortant showed more acute signs and earlier mortality than the RGNNV and SJNNV strains. Differences between strains were also observed in the possible portals of entry. Thus, whereas the reassortant strain could infect sole mainly through the skin or the oral route, and, to a minor extent, through the gills, the SJNNV strain seems to enter fish only through the gills and the RGNNV strain could use all tissues indistinctly. Taken together, all these results support the hypothesis that reassortment has improved betanodavirus infectivity for sole.

In vitro and in vivo characterization of molecular determinants of virulence in reassortant betanodavirus.

We previously reported that betanodavirus reassortant strains [redspotted grouper nervous necrosis virus/striped jack nervous necrosis virus (SJNNV)] isolated from Senegalese sole (Solea senegalensis) exhibited a modified SJNNV capsid amino acid sequence, with changes at aa 247 and 270. In the current study, we investigated the possible role of both residues as putative virulence determinants. Three recombinant viruses harbouring site-specific mutations in the capsid protein sequence, rSs160.03247 (S247A), rSs160.03270 (S270N) and rSs160.03247+270 (S247A/S270N), were generated using a reverse genetics system. These recombinant viruses were studied in cell culture and in vivo in the natural fish host. The three mutant viruses were shown to be infectious and able to replicate in E-11 cells, reaching final titres similar to the WT virus, although with a somewhat slower kinetics of replication. When the effect of the amino acid substitutions on virus pathogenicity was evaluated in Senegalese sole, typical clinical signs of betanodavirus infection were observed in all groups. However, fish mortality induced by all three mutant viruses was clearly affected. Roughly 40 % of the fish survived in these three groups in contrast with the WT virus which killed 100 % of the fish. These data demonstrated that aa 247 and 270 play a major role in betanodavirus virulence although when both mutated aa 247 and 270 are present, corresponding recombinant virus was not further attenuated.

Discussing the midwifery scope of practice within the NANDA-I taxonomy.

PURPOSE: To discuss the midwifery scope of practice within the NANDA-I taxonomy. METHODS: Review of the NANDA-I taxonomy followed by critical analysis of the nursing diagnoses (NDs) within the scope of midwifery practice. Search terms were defined. Two authors independently analyzed all diagnoses, and another author validated the results. FINDINGS: Of the 267 NDs in the NANDA-I taxonomy, 39 (14.6%) perceived a relationship with midwifery's scope of practice, and among these, 15 (5.6%) guaranteed greater specificity and accuracy for clinical reasoning. CONCLUSIONS: The suitability of NDs for the broad spectrum of the midwifery scope of practice may need to be completed. Women's responses to life events and health conditions are simultaneously complex and specific, and many NDs in the NANDA-I terminology may be far from fully identified and developed to represent these responses. IMPLICATIONS FOR NURSING PRACTICE: Improvements in NANDA-I diagnosis would contribute to advanced terminology and increased specificity and accuracy of the diagnostic process. This is important in achieving appropriate outcomes and safe and effective interventions for which the nurses and nurse-midwives are accountable. Many NDs would not necessarily be used in any other context since they are specific to midwifery; however, it would help to develop a NANDA-I taxonomy that is more inclusive and comprehensive internationally.

OBJETIVO: Discutir o âmbito da prática de atenção materna dentro da taxonomia da NANDA­I. MÉTODOS: Revisão da taxonomia da NANDA­I com análise crítica dos diagnósticos de enfermagem, no âmbito da prática de atenção materna. Os termos de pesquisa foram definidos. Dois autores analisaram, independentemente, todos os diagnósticos e um outro autor validou os resultados. RESULTADOS: Dos 267 diagnósticos de enfermagem da taxonomia NANDA­I, em 39 (14,6%) percebe­se relação com o âmbito da prática de atenção materna. Além disso, destes 38 diagnósticos incluídos, 15 (5,6%) garantem maior especificidade e acurácia para o raciocínio clínico. CONCLUSÕES: A adequação dos diagnósticos de enfermagem ao amplo espectro da prática de atenção materna pode precisar ser completada. As respostas das mulheres aos acontecimentos de vida e às condições de saúde são, simultaneamente, complexas e específicas e muitos diagnósticos de enfermagem na terminologia da NANDA­I podem não estar totalmente identificados e desenvolvidos para representar essas respostas. IMPLICAÇÕES PARA A PRÁTICA DE ENFERMAGEM: Melhorias nos diagnósticos da NANDA­I contribuiriam para uma terminologia avançada e para o aumento da especificidade e precisão do processo de diagnóstico. Isto é importante para a obtenção de resultados apropriados e intervenções seguras e eficazes, pelas quais os enfermeiros de atenção à saúde materna são responsáveis. Muitos diagnósticos de enfermagem não seriam necessariamente usados em qualquer outro contexto, uma vez que são específicos da atenção materna; no entanto, ajudaria a desenvolver uma taxonomia NANDA­I mais inclusiva e abrangente, a nível internacional.

A Potential Nervous Necrosis Virus (NNV) Live Vaccine for Sole Obtained by Genomic Modification.

Viral Encephalopathy and Retinopathy (VER) is a neurological infectious fish disease that causes vacuolization and necrosis in the central nervous system, which lead to swimming abnormalities and, generally, host death in the early stages of development. VER is caused by the Nervous Necrosis Virus (NNV), a non-enveloped virus with a bisegmented and positive-stranded (+) RNA genome. The largest segment (RNA1) codes for viral polymerase while capsid protein is encoded by RNA2. The aim of this study was to explore the potential of a reverse-engineered RGNNV/SJNNV strain that harbors mutations in both 3'NCRs (position 3073 of RNA1 and 1408 and 1412 of RNA2) as an attenuated live vaccine for sole. The attenuation of this strain was confirmed through experimental infections in sole at 22 °C. Vaccination trials were performed by bath, intramuscular, and intraperitoneal injection, at two temperatures (18 and 22 °C). Our results indicate the improved survival of vaccinated fish and delayed and poorer viral replication, as well as an overexpression of immune response genes linked to T cell markers (cd4 and cd8), to an early inflammatory response (tlr7 and tnfα), and to antiviral activity (rtp3 and mx). In conclusion, our study indicates that the attenuated strain is a good vaccine candidate as it favors sole survival upon infection with the wt strain while inducing a significant immune response.

Women's fear of childbirth during pregnancy: A concept analysis.

PURPOSE: To map and analyze the concept of women's fear of childbirth (FOC) during pregnancy and to develop a new nursing diagnosis (ND). METHODS: Concept analysis based on Walker and Avant's methodology. An integrative literature search was conducted in Scopus, Web of Science, and CINAHL Complete databases, including peer-reviewed articles published in English from January 1981 to October 2022. FINDINGS: Thirty-one studies were included in the review. Women's FOC during pregnancy comprises three central categories: attributes, antecedents, and consequences. Nine main characteristics (attributes) are related to one or more of eight prior conditions (antecedents) that have the potential to exert negative or favorable impacts (consequences), depending on whether coping strategies have been a part of a fearful woman's pregnancy. These elements and relationships among them should be viewed as dynamic. CONCLUSIONS: Women's FOC during pregnancy is a subjective, multidimensional, and dynamic concept that benefits prenatal care responses. A conceptual matrix on prenatal care will consider the factors underlying women's FOC and their potential mechanisms, how fearful pregnant women respond, the aspects that shape FOC, and how it influences outcomes when supporting pregnant women with FOC. Therefore, the study findings can be used to inform ways of identifying fearful women or as a basis for interventions to manage fears and enhance the confidence of pregnant women for childbirth. IMPLICATIONS FOR NURSING PRACTICE: The results of this study help nurses, OB nursing practitioners, nurse-midwives, and midwives to identify the condition of women's FOC during pregnancy and guide nursing therapeutic interventions to decrease the negative effects on women's health and well-being. Therefore, there are valuable reasons to propose using "fear of childbirth" as a ND in the class "coping responses" within domain coping/stress tolerance, contributing to refining the NANDA-I taxonomy.

OBJETIVO: Mapear e analisar o conceito de medo do parto em mulheres durante a gravidez e desenvolver um novo diagnóstico de enfermagem. MÉTODOS: Análise de conceito com base na metodologia de Walker e Avant. Foi realizada uma pesquisa integrativa da literatura nas bases de dados Scopus, Web of Science e CINAHL Complete e incluiu artigos revistos por pares, publicados em inglês, de janeiro de 1981 a outubro de 2022. RESULTADOS: Trinta e um estudos foram incluídos na revisão. O medo do parto em mulheres durante a gravidez é composto por três categorias centrais: atributos, antecedentes e consequências. Nove características principais (atributos) estão relacionadas com uma ou mais das oito condições anteriores (antecedentes) que têm o potencial de exercer um impacto negativo ou favorável (consequências), dependendo se estratégias de coping fizeram parte da gravidez de uma mulher com medo. Estes elementos e as relações entre eles devem ser vistos como dinâmicos. CONCLUSÕES: O medo do parto em mulheres durante a gravidez é um conceito subjetivo, multidimensional e dinâmico que beneficia de respostas pelos cuidados pré-natais. Uma matriz conceptual sobre cuidados pré-natais considerará os fatores subjacentes ao medo do parto nas mulheres e os seus potenciais mecanismos, como as mulheres grávidas com medo respondem, os aspetos que moldam o medo do parto, e como influenciam os resultados quando as mulheres grávidas com medo do parto são apoiadas. Portanto, os resultados do estudo podem ser usados para identificar mulheres com medo ou desenvolver intervenções que permitam gerir os medos e aumentar a confiança das mulheres grávidas para o parto. IMPLICAÇÕES PARA A PRÁTICA DE ENFERMAGEM: Os resultados deste estudo ajudarão os enfermeiros de atenção à saúde materna a identificar a condição do medo do parto nas mulheres durante a gravidez e orientar intervenções terapêuticas de enfermagem, no sentido de diminuir os efeitos negativos do medo na saúde e no bem-estar das mulheres. Portanto, há razões relevantes para propor a utilização do "medo do parto" como um diagnóstico de enfermagem na classe "coping responses" do domínio "coping/stress tolerance", contribuindo para o refinamento da taxonomia NANDA-I.

Designing and Validation of a Droplet Digital PCR Procedure for Diagnosis and Accurate Quantification of Nervous Necrosis Virus in the Mediterranean Area.

The viral nervous necrosis virus (VNNV) is the causative agent of an important disease affecting fish species cultured worldwide. Early and accurate diagnosis is, at present, the most effective control and prevention tool, and molecular techniques have been strongly introduced and accepted by official organizations. Among those, real-time quantitative polymerase chain reaction (rt-qPCR) is nowadays displacing other molecular techniques. However, another PCR-based technology, droplet digital PCR (ddPCR), is on the increase. It has many advantages over qPCR, such as higher sensitivity and more reliability of the quantification. Therefore, we decided to design and validate a protocol for the diagnosis and quantification of SJ and RG type VNNV using reverse transcription-ddPCR (RT-ddPCR). We obtained an extremely low limit of detection, 10- to 100-fold lower than with RT-qPCR. Quantification by RT-ddPCR, with a dynamic range of 6.8-6.8 × 104 (SJ type) or 1.04 × 101-1.04 × 105 (RG type) cps/rctn, was more reliable than with RT-qPCR. The procedure was tested and validated in field samples, providing high clinical sensitivity and negative predictive values. In conclusion, we propose this method to substitute RT-qPCR protocols because it exceeds the expectations of qPCR in the diagnosis and quantification of VNNV.

Recombinant viral hemorrhagic septicemia virus with rearranged genomes as vaccine vectors to protect against lethal betanodavirus infection.

The outbreaks of viral hemorrhagic septicemia (VHS) and viral encephalopathy and retinopathy (VER) caused by the enveloped novirhabdovirus VHSV, and the non-enveloped betanodavirus nervous necrosis virus (NNV), respectively, represent two of the main viral infectious threats for aquaculture worldwide. Non-segmented negative-strand RNA viruses such as VHSV are subject to a transcription gradient dictated by the order of the genes in their genomes. With the goal of developing a bivalent vaccine against VHSV and NNV infection, the genome of VHSV has been engineered to modify the gene order and to introduce an expression cassette encoding the major protective antigen domain of NNV capsid protein. The NNV Linker-P specific domain was duplicated and fused to the signal peptide (SP) and the transmembrane domain (TM) derived from novirhabdovirus glycoprotein to obtain expression of antigen at the surface of infected cells and its incorporation into viral particles. By reverse genetics, eight recombinant VHSVs (rVHSV), termed NxGyCz according to the respective positions of the genes encoding the nucleoprotein (N) and glycoprotein (G) as well as the expression cassette (C) along the genome, have been successfully recovered. All rVHSVs have been fully characterized in vitro for NNV epitope expression in fish cells and incorporation into VHSV virions. Safety, immunogenicity and protective efficacy of rVHSVs has been tested in vivo in trout (Oncorhynchus mykiss) and sole (Solea senegalensis). Following bath immersion administration of the various rVHSVs to juvenile trout, some of the rVHSVs were attenuated and protective against a lethal VHSV challenge. Results indicate that rVHSV N2G1C4 is safe and protective against VHSV challenge in trout. In parallel, juvenile sole were injected with rVHSVs and challenged with NNV. The rVHSV N2G1C4 is also safe, immunogenic and efficiently protects sole against a lethal NNV challenge, thus presenting a promising starting point for the development of a bivalent live attenuated vaccine candidate for the protection of these two commercially valuable fish species against two major diseases in aquaculture.

Prevalence and predictive factors for fear of childbirth in pregnant Portuguese women: A cross-sectional study.

OBJECTIVE: To measure the prevalence of the fear of childbirth (FOC) and determine which factors predict severe FOC among pregnant Portuguese women. METHODS: An online cross-sectional study among pregnant Portuguese women aged ≥ 20 years who were recorded using a convenience sampling. Self-administered questionnaires were used for data collection: socio-demographic and obstetric questionnaire and European Portuguese version of Wijma Delivery Expectancy Questionnaire-version A (WDEQ-A). Data of 669 participants were collected successfully from June 9 to October 30, 2019. Predictive factors for severe FOC were investigated using a multivariate logistic regression analysis. Odds ratios (OR) and 95% confidence intervals were calculated. RESULTS: The prevalence of severe FOC (WDEQ-A ≥ 85) among pregnant Portuguese women was 10%. Severe FOC was significantly associated with lower educational level, single/divorced marital status, and negative previous childbirth experience. Multivariate logistic regression analysis indicated that being single or divorced and having a negative previous childbirth experience were predictive variables for severe FOC. CONCLUSION: Pregnant Portuguese women have FOC, although with varying severity. The data suggest that marital status and women's perceptions of previous childbirth experience may be useful variables to predict severe FOC. Further research for extending the predictive factors of FOC should be refined. The results are clinically relevant for midwifery care, as they should be used in the sense of early identification of fearful pregnant women to provide adequate support strategies to reduce FOC.

Midwives' interventions for reducing fear of childbirth in pregnant women: a scoping review.

OBJECTIVE: The objective of this review was to map and analyze midwives' interventions for reducing fear of childbirth in pregnant women. INTRODUCTION: Fear of childbirth is a phenomenon negatively affecting women's health and well-being before and during pregnancy. Over the past few decades, there has been growing research interest in interventions for reducing fear of childbirth in pregnant women. One of the challenges in midwifery care is to provide an appropriate model of care for pregnant women with fear of childbirth. Further research efforts are needed to identify midwives' interventions for reducing fear of childbirth in pregnant women and to examine their characteristics. INCLUSION CRITERIA: This scoping review considered studies that included midwives' interventions for reducing fear of childbirth in pregnant women. Specifically, interventions were led and/or implemented by midwives during the antenatal period, and integrating all possible midwifery practice settings. Quantitative, qualitative, and mixed methods studies were included. This review also considered systematic reviews, text and opinion papers, and conference abstracts. METHODS: The JBI methodology for conducting scoping reviews was used. Published and unpublished literature in English, Portuguese, and Spanish from January 1981 to October 2020 was included. MEDLINE (PubMed), CINAHL Complete, APA PsycINFO, Scopus, Embase, Web of Science, SciELO, MedicLatina, Academic Search Complete, ERIC, Psychology and Behavioral Sciences Collection, and the Cochrane Library databases were searched. Searches for gray literature were also undertaken on the Repositório Científico de Acesso Aberto de Portugal, ProQuest Dissertations and Theses, British Library EThOS, OvidSP Resource Center, Banco de Teses da CAPES, and OpenGrey. A three-step search strategy was followed, and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis extension for Scoping Reviews checklist was used. Two independent reviewers extracted the data using a data extraction tool developed specifically for this scoping review. RESULTS: A total of 3704 articles were identified and screened, of which 34 articles were included. The majority of studies had been published in the past 10 years (88%) in Scandinavian countries or Australia (79%). Several midwives' antenatal interventions were found, such as midwife-led team models of care. Midwives played a facilitator role that varied across the included studies. In 20 studies (59%), midwives led and implemented the interventions alone ( n   =  13; 38%) or with the participation of other health professionals ( n   =  7; 21%). In the remaining 14 studies (41%), midwives were part of a multidisciplinary team that included different health professionals (mainly obstetricians and psychologists) who had been involved in delivering interventions alongside midwives or with minor participation from midwives. Counseling ( n   =  12; 35%) and psychoeducation ( n   =  8; 24%) were the most common midwife interventions for reducing fear of childbirth in pregnant women. CONCLUSIONS: Midwives working across their full scope of practice play a pivotal role in reducing fear of childbirth, which may explain the variety of midwives' antenatal interventions. Reducing fear of childbirth in pregnant women and promoting normal childbirth as a positive experience are key features of midwives' interventions, which should include women's empowerment measures. Evidence-based midwife-led intervention programs for pregnant women with fear of childbirth should be designed and tested to improve clinical practice as well as women's reproductive outcomes and perinatal experiences.

Nervous Necrosis Virus (NNV) Booster Vaccination Increases Senegalese Sole Survival and Enhances Immunoprotection.

A re-immunization programme has been tested to improve the protective response elicited in sole by a previously developed BEI-inactivated betanodavirus vaccine. The vaccine was prepared using a reassortant RGNNV/SJNNV strain which is highly pathogenic for sole, and vaccination assays were performed by intraperitoneal injection. Experimental design included a prime- and a booster-vaccination group, which consisted of individuals that received a second vaccine injection at 30 days post vaccination), and their respective controls. A month after prime/booster vaccination, fish were challenged by intramuscular injection with the homologous NNV strain. Samples were collected at different times post vaccination and post challenge to assess the immune response and viral replication. Booster dose enhanced the protection against NNV infection because a significant increase in survival was recorded when compared with prime-vaccinated individuals (relative percent survival 77 vs. 55). In addition, a clear decrease in viral replication in the brain of challenged sole was observed. During the immune induction period, no differences in IgM production were observed between prime- and booster-vaccinated fish, and the expression of the antigen presenting cells (APC)-related molecule MHC class II antigen was the only differential stimulation recorded in the re-immunized individuals. However, a significant upregulation of mhcII and the lymphocytes T helper (Th) marker cd4 was observed after the challenge in the booster-vaccinated group, suggesting these cells play a role in the protection conferred by the booster injection. In addition, after viral infection, re-immunized fish showed specific and neutralizing antibody production and overexpression of other immune-related genes putatively involved in the control of NNV replication.

Development and Validation of a SYBR Green Real Time PCR Protocol for Detection and Quantification of Nervous Necrosis Virus (NNV) Using Different Standards.

The nervous necrosis virus (NNV) is a threat to fish aquaculture worldwide, especially in Mediterranean countries. Fast and accurate diagnosis is essential to control it, and viral quantification is required to predict the level of risk of new viral detections in field samples. For both, reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) is used by diagnostic laboratories. In the present study, we developed an RT-qPCR procedure for the diagnosis and simultaneous quantification of NNV isolates from any of the four genotypes. The method proved to be highly sensitive in terms of crude virus titer: 5.56-9.88 TCID50/mL (tissue culture infectious dose per mL), depending on the viral strain, and averaging 8.8 TCID50/mL or 0.08 TCID50/reaction. Other standards also yielded very low detection limits: 16.3 genome copies (cps) of purified virus per mL, 2.36 plasmid cps/mL, 7.86 in vitro synthetized RNA cps/mL, and 3.16 TCID50/mL of virus from infected tissues. The diagnostic parameters evaluated in fish samples were much higher in comparison to cell culture isolation and nested PCR. In addition, the high repeatability and reproducibility of the procedure, as well as the high coefficient of determination (R2) of all the calibration curves with any type of standard tested, ensure the high reliability of the quantification of NNV using this RT-qPCR procedure, regardless of the viral type detected and from the type of standard chosen.

Differential Nervous Necrosis Virus (NNV) Replication in Five Putative Susceptible Cell Lines.

Viral encephalopathy and retinopathy caused by nervous necrosis virus (NNV), is one of the most threatening viral diseases affecting marine fish worldwide. In vitro propagation of NNV strains is essential for the design of effective control measures. In the present study we analysed both the susceptibility and the permissiveness of five fish cell lines (E-11, GF-1, SAF-1, DLB-1, and SaB-1) to three NNV strains (one RGNNV, one SJNNV, and one reassortant RGNNV/SJNNV). E-11 and DLB-1 were demonstrated to be highly susceptible to NNV strains, with average adsorption efficiency (AE) values higher than 90%. SAF-1 also showed high susceptibility (AE 88%), whereas GF-1 can be regarded as moderately susceptible (AE around 50%). On the contrary, SaB-1 can be considered a poorly susceptible cell line (AE values below 20%). E-11 and GF-1 cell lines provided the highest production rates for RGNNV and RG/SJ (around 103) and both cell lines can be regarded as fully permissive for these viral types. However, the SJNNV production rate in GF-1 was only 17.8 and therefore this cell line should be considered semi-permissive for this genotype. In SAF-1 cells, moderate viral replication was recorded but differences in intracellular and extracellular production suggest that viral progeny was not efficiently released. In DLB-1 and SaB-1 the final viral titres obtained in E-11 were lower than those of the inoculum. However, RNA1 synthesis values seem to indicate that RGNNV replication in DLB-1 and SAF-1 could have been underestimated, probably due to a poor adaptation of the virus grown in these cell lines to E-11. Based on all these results, E-11 seems to be the most appropriate cell for in vitro culture of RGNNV, SJNNV, and reassortant strains.