Production and characterization of egg yolk antibodies against the ZIKV NS2B expressed in Nicotiana benthamiana.

The emergence of Zika virus (ZIKV) and its associated neonatal and congenital complications pose a threat to global health, particularly in tropical and subtropical regions with co-circulation of related flaviviruses and intense vector proliferation. Diagnosis of ZIKV by RT-PCR is limited to the viraemic phase and is not always accessible in low-income tropical settings, while serological tests often show cross-reactivity with other flaviviruses. Given the similarity of ZIKV symptoms to those of other arboviruses, but the different prognosis and risks, it is important to develop specific and accessible diagnostic tools. Egg yolk antibodies (IgY) were obtained from Leghorn laying hens immunized with recombinant ZIKV NS2B protein produced in agroinfiltrated Nicotiana benthamiana. After three immunizations, total IgY was recovered from the eggs by the 20% ammonium sulfate precipitation method. After characterisation by SDS-PAGE, dot blotting and ELISA, the IgY was adsorbed to dengue virus (DENV) from cell culture supernatants and tested for its ability to specifically detect ZIKV-positive sera samples. High yield and purity were observed on SDS-PAGE for polyclonal IgY, which reacted with NS2B at high titres in ELISA and detected both NS2B and ZIKV in dot blotting. However, a cross-reaction with DENV was observed and the anti-NS2B IgY was unable to discriminate ZIKV from DENV positive sera samples, even after adsorption with DENV. This is probably due to the phylogenetic relationship of the viruses and the shared identity of their proteins.

Natural versus recombinant viral antigens in SARS-COV-2 serology: challenges in optimizing laboratory diagnosis of COVID19

OBJECTIVES: COVID-19 is a public health emergency of international concern whose detection in recovered asymptomatic patients is dependent on accurate diagnosis as it enables the estimation of the susceptibility of the population to the infection. This demand has resulted in the development of several commercial assays employing recombinant proteins, but the results of these assays are not reliable as they do not involve comparison with natural viral antigens. We independently used the SARS-CoV-2 whole viral antigen (WVA) and recombinant nucleocapsid protein (rNP) to develop in-house ELISAs for IgG detection; the results of these ELISAs were then compared to obtain reliable results. METHODS: WVA and rNP ELISAs were performed on COVID-19 negative sera from patients before the pandemic in Brazil, and on RT-qPCR-positive or SARS-CoV-2-IgG against rNP and IgG against WVA-positive samples from recently infected patients in Sao Paulo, Brazil. RESULTS: Both ELISAs detected a large fraction of infected patients but exhibited certain drawbacks. Higher signals and lower numbers of false-negatives were observed in rNP ELISA; however, a higher fraction of false-positives was observed in control groups. A high number of false-negatives was observed with WVA ELISA. Correlating the results of rNP and WVA ELISAs resulted in improved performance for COVID-19 diagnosis. CONCLUSION: The choice of antigen is an important aspect in optimizing the laboratory diagnosis of COVID-19. The use of rNP ELISA for the detection of anti-SARS-CoV-2 IgG antibodies seems promising, but comparison of the results with those of WVA ELISA is crucial for accurate test development prior to commercialization. IgG serology using several assays, and with the spectral patterns of SARS-CoV-2, resulted in confusing information that must be clarified before the establishment of diagnostic serology criteria.

Chronic hepatitis B: The interplay between intrahepatic lymphocyte population and viral antigens in relation to liver damage.

In Chronic hepatitis B (CHB) infection, virus and immune response interplay is thought to be responsible for pathogenesis. Yet, the impact of each immune cell population and viral protein expression in liver damage is still unknown. Our aim was to study the interplay between intrahepatic immune response and viral activity in relation to CHB liver damage. Immunostaining was performed in 29 liver biopsies from untreated CHB patients to characterize liver infiltrate [Th (CD4+), CTL (CD8+), Treg (FoxP3+), Th17 (IL-17A+) and Th1 (T-bet+)] and viral antigen expression (HBsAg and HBcAg). Inflammatory activity and fibrosis were assessed using the HAI and METAVIR scoring system. All studied populations were identified in the portal-periportal (P-P) areas with a CD4+ lymphocyte predominance, while only CD8+ and FoxP3+ cells were observed in the intralobular area. Both P-P CD4+ and intralobular CD8+ cell frequencies were increased among severe hepatitis cases. Concerning HBsAg and HBcAg expression, a mutually exclusive pattern was observed. HBcAg was mainly detected among HBeAg-positive patients and was associated with hepatitis severity and higher frequency of P-P FoxP3+, intralobular CD8+ and FoxP3+ cells. HBsAg was identified among HBeAg-negative cases with less severe hepatitis grade and lower frequency of P-P CD4+ and intralobular FoxP3+ lymphocytes. In conclusion, the HBV antigen profile expression seen during CHB infection may be reflecting different stages of viral replication which impacts the host immune response and liver damage process. While HBcAg might be an inducer of a regulatory microenvironment, the intralobular CTL population seemed to have a key role in hepatitis severity.

Pesquisa de antígenos e anticorpos contra Circovírus Suíno II em suínos com e sem sintomatologia da síndrome multisistêmica do definhamento em granjas comerciais mineiras / Research of antigen and antibodies against Porcine Circovirus Type - 2 in pigs with and without postweaning multsystemic wasting syndrome from commercial farms of Minas Gerais state

Circovirus Suíno Tipo-2 (CSV-2) é um vírus não-envelopado, DNA fita única circular, classificado na família de Circoviridae, relacionado à Síndrome Multisistêmica do Definhamento de Suínos (SMDS) de leitões. A técnica de Imuno-Histoquímica (IHQ) é aplicada na detecção de antígenos do (CSV-2) em tecido lesionados. Entretanto, suínos infectados com CSV-2 assintomáticos ou com infecções subclínicas podem disseminar o vírus no plantel. A pesquisa de anticorpos contra o CVS-2 em porcos aparentemente saudáveis pode sugerir o risco de ingresso de vírus. Neste trabalho foram investigados antígenos e anticorpos contra CVS-2 de porcos de granjas comerciais de sete e oito mesorregiões de Estado de Gerais de Minas (MG). 32 porcos com idades de cinco a onze semanas que apresentaram sinais clínicos de SMDS foram necropsiados. Antígenos do CVS-2 foram investigados nos porcos sacrificados (grupo I) e amostras de demanda de diagnóstico (grupo II) pela IHQ. 7.60% e mais de 60%do primeiro e segundo grupo, respectivamente, foram positivos para antígeno viral. Em todos, marcação intensa de macrófagos e histiócitos, especialmente nos linfonodos e pulmão, evidenciou antígenos a CVS-2. Em paralelo, 955 amostras de soros de porcos de 35 granjas comerciais de ciclo completo sem sintomatologia clínica de SMDS foram investigadas para a detecção de anticorpos contra o CVS-2 aplicando a técnica de Imunoperoxidase em Monocamada de Células (IPMC). Todos os rebanhos de suínos (100%) apresentaram animais positivos (nível de confiança 90% a 100%) com freqüência de 96,6% de porcos reagentes (nível de confiança 94.7% a 98,6%). Os títulos de anticorpos contra o CVS-2 variou 1:320 (médio) a 1:10.240 (alto). Os resultados sugerem que pelo menos 2,66% e 9% dos porcos das regiões Triângulo Mineiro e Zona da Mata, respectivamente, nesta amostragem poderiam desenvolver SMDS clínica e esta porcentagem alcança 3,35% no total dos soros.

Porcine Circovirus Type 2 (PCV-2) is a non-enveloped circular single stranded DNA virus classified in the Circoviridae family related to post weaning multi systemic wasting syndrome (PMWS) in piglets.Immune-Histochemical (IHC) techniques are applied to detected PCV-2 antigen in the animal tissue injuries. Although, asymptomatic or sub clinic PCV-2 infected pigs could disseminate the virus in the flock. Serologic survey on apparently health pigs could suggest the virus ingression risk. In this work, antigens and antibodies against PCVS-2 in swine from commercial farms of seven and eight mesorregions of Minas Gerais State (MG) were investigated. 32 pigs with ages from five to eleven weeks which presented SMDS clinical signs were submitted to necropsy. PCV-2 antigens were investigated either from sacrificed pigs (Group I) and diagnosis demand samples (Group II) by IHC. 7,60% and more than 60% of the first and second groups, respectively, were positive for viral antigen. In both of them, intense marking of macrophages and histiocytes, especially in the lymph nodes and lung, evidenced antigens to CVS-2. In parallel, Immunoperoxidase Monolayer Assay (IPMA) was applied to antibody against PCV-2 screened in 955 pigs from 35 complete cycle commercial farms from same mesorregions. All pig flocks (100%) presented positive animals (confidence level 90% to 100%) and the frequency of reacting pigs varied 96.6% (confidence level 94,7% to 98,6%). PCV-2 antibody titers ranged 1:320 (medium) to 1:10.240 (high). The results suggest that 2.66% and 9% of pigs from Triângulo Mineiro and Zona da Mata regions respectively, would be able to develop clinical SMDS and that percentage reach 3.35% in the total serum.

Research of antigen and antibodies against Porcine Circovirus Type - 2 in pigs with and without postweaning multsystemic wasting syndrome from commercial farms of Minas Gerais State / Pesquisa de antígenos e anticorpos contra Circovírus Suíno II em suínos com e sem sintomatologia da síndrome multisistêmica do definhamento em granjas comerciais mineiras

Porcine Circovirus Type 2 (PCV-2) is a non-enveloped circular single stranded DNA virus classified in the Circoviridae family related to post weaning multi systemic wasting syndrome (PMWS) in piglets. Immune-Histochemical (IHC) techniques are applied to detected PCV-2 antigen in the animal tissue injuries. Although, asymptomatic or sub clinic PCV-2 infected pigs could disseminate the virus in the flock. Serologic survey on apparently health pigs could suggest the virus ingression risk. In this work, antigens and antibodies against PCVS-2 in swine from commercial farms of seven and eight mesorregions of Minas Gerais State (MG) were investigated. 32 pigs with ages from five to eleven weeks which presented SMDS clinical signs were submitted to necropsy. PCV-2 antigens were investigated either from sacrificed pigs (Group I) and diagnosis demand samples (Group II) by IHC. 7,60% and more than 60% of the first and second groups, respectively, were positive for viral antigen. In both of them, intense marking of macrophages and histiocytes, especially in the lymph nodes and lung, evidenced antigens to CVS-2. In parallel, Immunoperoxidase Monolayer Assay (IPMA) was applied to antibody against PCV-2 screened in 955 pigs from 35 complete cycle commercial farms from same mesorregions. All pig flocks (100%) presented positive animals (confidence level 90% to 100%) and the frequency of reacti

Circovirus Suíno Tipo-2 (CSV-2) é um vírus não-envelopado, DNA fita única circular, classificado na família de Circoviridae, relacionado à Síndrome Multisistêmica do Definhamento de Suínos (SMDS) de leitões. A técnica de Imuno-Histoquímica (IHQ) é aplicada na detecção de antígenos do (CSV-2) em tecido lesionados. Entretanto, suínos infectados com CSV-2 assintomáticos ou com infecções subclínicas podem disseminar o vírus no plantel. A pesquisa de anticorpos contra o CVS-2 em porcos aparentemente saudáveis pode sugerir o risco de ingresso de vírus. Neste trabalho foram investigados antígenos e anticorpos contra CVS-2 de porcos de granjas comerciais de sete e oito mesorregiões de Estado de Gerais de Minas (MG). 32 porcos com idades de cinco a onze semanas que apresentaram sinais clínicos de SMDS foram necropsiados. Antígenos do CVS-2 foram investigados nos porcos sacrificados (grupo I) e amostras de demanda de diagnóstico (grupo II) pela IHQ. 7.60% e mais de 60%do primeiro e segundo grupo, respectivamente, foram positivos para antígeno viral. Em todos, marcação intense de macrófagos e histiócitos, especialmente nos linfonodos e pulmão, evidenciou antígenos a CVS-2. Em paralelo, 955 amostras de soros de porcos de 35 granjas comerciais de ciclo completo sem sintomatologia clínica de SMDS foram investigadas para a detecção de anticorpos contra o CVS-2 aplicando a técnica de Imunopero