Prediction of the antigenic regions in eight RhD variants identified by computational biology.

BACKGROUND AND OBJECTIVES: Changes in RHD generate variations in protein structure that lead to antigenic variants. The classical model divides them into quantitative (weak and Del) and qualitative (partial D). There are two types of protein antigens: linear and conformational. Computational biology analyses the theoretical assembly of tertiary protein structures and allows us to identify the 'topological' differences between isoforms. Our aim was to determine the theoretical antigenic differences between weak RhD variants compared with normal RhD based on structural analysis using bioinformatic techniques. MATERIALS AND METHODS: We analysed the variations in secondary structures and hydrophobicity of RHD*01, RHD*01W.1, W2, W3, RHD*09.03.01, RHD*09.04, RHD*11, RHD*15 and RHD*21. We then modelled the tertiary structure and calculated their probable antigenic regions, intra-protein interactions, displacement and membrane width and compared them with Rhce. RESULTS: The 10 proteins are similar in their secondary structure and hydrophobicity, with the main differences observed in the exofacial coils. We identified six potential antigenic regions: one that is unique to RhD (R3), one that is common to all D (R6), three that are highly variable among RhD isoforms (R1, R2 and R4), one that they share with Rhce (R5) and two that are unique to Rhce (Ra and Rbc). CONCLUSION: The alloimmunization capacity of these subjects could be explained by the variability of the antigen pattern, which is not necessarily recognized or recognized with lower intensity by the commercially available antibodies, and not because they have a lower protein concentration in the membrane.

Development of an accurate and rapid method for whole genome characterization of canine parvovirus.

Canine parvovirus is a highly contagious pathogen affecting domestic dogs and other carnivores globally. Monitoring CPV through continuous genomic surveillance is crucial for mapping variability and developing effective control measures. Here, we developed a method using multiplex-PCR-next-generation sequencing to obtain full-length CPV genomes directly from clinical samples. This approach utilizes tiling and tailed amplicons to amplify overlapping fragments of roughly 250 base pairs. This enables the creation of Illumina libraries by conducting two PCR reaction runs. We tested the assay in 10 fecal samples from dogs diagnosed with CPV and one CPV-2 vaccine strain. Furthermore, we applied it to a feline sample previously diagnosed with the feline panleukopenia virus. The assay provided 100 % genome coverage and high sequencing depth across all 12 samples. It successfully provided the sequence of the coding regions and the left and right non-translated regions, including tandem and terminal repeats. The assay effectively amplified viral variants from divergent evolutionary groups, including the antigenic variants (2a, 2b, and 2c) and the ancestral CPV-2 strain included in vaccine formulations. Moreover, it successfully amplified the entire genome of the feline panleukopenia virus found in cat feces. This method is cost-effective, time-efficient, and does not require lab expertise in Illumina library preparation. The multiplex-PCR-next-generation methodology facilitates large-scale genomic sequencing, expanding the limited number of complete genomes currently available in databases and enabling real-time genomic surveillance. Furthermore, the method helps identify and track emerging CPV viral variants, facilitating molecular epidemiology and control. Adopting this approach can enhance our understanding of the evolution and genetic diversity of Protoparvovirus carnivoran1.

Sequence-Based Antigenic Analyses of H1 Swine Influenza A Viruses from Colombia (2008-2021) Reveals Temporal and Geographical Antigenic Variations.

Swine influenza is a respiratory disease that affects the pork industry and is a public health threat. It is caused by type A influenza virus (FLUAV), which continuously undergoes genetic and antigenic variations. A large amount of information regarding FLUAV in pigs is available worldwide, but it is limited in Latin America. The HA sequences of H1 subtype FLUAV-positive samples obtained from pigs in Colombia between 2008-2021 were analyzed using sequence-based antigenic cartography and N-Glycosylation analyses. Of the 12 predicted global antigenic groups, Colombia contained five: four corresponding to pandemic strains and one to the classical swine H1N1 clade. Circulation of these clusters was observed in some regions during specific years. Ca2 was the immunodominant epitope among Colombian viruses. The counts of N-Glycosylation motifs were associated with the antigenic cluster ranging from three to five. The results show for the first time the existence of antigenic diversity of FLUAV in Colombia and highlight the impact of spatial and temporal factors on this diversity. This study provides information about FLUAV variability in pigs under natural conditions in the absence of vaccination and emphasizes the need for surveillance of its phylogenetic and antigenic characteristics.

Antibodies to variable surface antigens induce antigenic variation in the intestinal parasite Giardia lamblia.

The genomes of most protozoa encode families of variant surface antigens. In some parasitic microorganisms, it has been demonstrated that mutually exclusive changes in the expression of these antigens allow parasites to evade the host's immune response. It is widely assumed that antigenic variation in protozoan parasites is accomplished by the spontaneous appearance within the population of cells expressing antigenic variants that escape antibody-mediated cytotoxicity. Here we show, both in vitro and in animal infections, that antibodies to Variant-specific Surface Proteins (VSPs) of the intestinal parasite Giardia lamblia are not cytotoxic, inducing instead VSP clustering into liquid-ordered phase membrane microdomains that trigger a massive release of microvesicles carrying the original VSP and switch in expression to different VSPs by a calcium-dependent mechanism. This novel mechanism of surface antigen clearance throughout its release into microvesicles coupled to the stochastic induction of new phenotypic variants not only changes current paradigms of antigenic switching but also provides a new framework for understanding the course of protozoan infections as a host/parasite adaptive process.

A(H3N2) antigenic variation of influenza is associated with low vaccine efficacy in the early 2018 influenza season in Mexico City.

OBJECTIVES: We evaluated the VE and the mutations of the viruses present in the Mexican population at the beginning of 2018. METHODS: We diagnosed influenza in outpatients with a high-performance Rapid Influenza Diagnostic Test (RIDT) qRT-PCR. Descriptive statistics were used to describe the study population, while the chi-square test was used to determine clinical variables. VE was analyzed through a negative test design. We sequenced the hemagglutinin (HA) gene, performed a phylogenetic analysis, and analyzed the nonsynonymous substitutions both in and outside antigenic sites. RESULTS: Of the 240 patients analyzed, 42.5% received the trivalent vaccine, and 37.5% were positive for influenza. The VE for the general population for any influenza virus type or subtype was 37.0%, while the VE for the predominant influenza A(H3N2) subtype was the lowest (19.7%). The phylogenetic analysis of HA showed the co-circulation of clades and subclades 3C.2a1, 3C.2a1b, 3C.2a2, 3C.2a2re, 3C.2a3, and 3C.3a with identities approximately 97-98% similar to the vaccine composition. CONCLUSION: Low VE was related to the co-circulation of multiple clades and subclades of influenza A(H3N2), with sufficient genetic and phenotypic distance to allow for the infection of vaccinated individuals.

Comprehensive characterization of Cysteine-rich protein-coding genes of Giardia lamblia and their role during antigenic variation.

Giardia lamblia encodes several families of cysteine-rich proteins, including the Variant-specific Surface Proteins (VSPs) involved in the process of antigenic variation. Their characteristics, definition and relationships are still controversial. An exhaustive analysis of the Cys-rich families including organization, features, evolution and levels of expression was performed, by combining pattern searches and predictions with massive sequencing techniques. Thus, a new classification for Cys-rich proteins, genes and pseudogenes that better describes their involvement in Giardia's biology is presented. Moreover, three novel characteristics exclusive to the VSP genes, comprising an Initiator element/Kozak-like sequence, an extended polyadenylation signal and a unique pattern of mutually exclusive transcript accumulation are presented, as well as the finding that High Cysteine Membrane Proteins, upregulated under stress, may protect the parasite during VSP switching. These results allow better interpretation of previous reports providing the basis for further studies of the biology of this early-branching eukaryote.

Antigenic diversity of MASP gene family of Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease (CD), is a heterogeneous species with high genetic and phenotypic diversity. MASP is the second largest multigene family of T. cruzi. The high degree of polymorphism of the family associated with its location at the surface of infective forms of T. cruzi suggests that MASP participates in mechanisms of host-parasite interaction. In this work, MASP members were divided into 7 subgroups based on protein sequence similarity, and one representative member from each subgroup was chosen to be expressed recombinantly. Immunogenicity of recombinant MASP proteins (rMASP) was investigated using different sera panels from T. cruzi infected mice. To mimic a natural condition in which different MASP members are expressed at the same time in the parasite population, a multiplex bead-based flow cytometry assay was also standardized. Results showed that rMASPs are poorly recognized by sera from mice infected with Colombiana strain, whereas sera from mice infected with CL Brener and Y display high reactivity against the majority of rMASPs tested. Flow cytometry showed that MASP recognition profile changes 10 days after infection. Also, multiplex assay suggests that MASP M1 and M2 are more immunogenic than the other MASP members evaluated that may play an immunodominant role during infection.

Early Thymectomy Is Associated With Long-Term Impairment of the Immune System: A Systematic Review.

Background and Aims: Congenital heart diseases (CHDs) are diagnosed in approximately 9 in 1,000 newborns, and early cardiac corrective surgery often requires partial or complete thymectomy. As the long-term effect of early thymectomy on the subsequent development of the immune system in humans has not been completely elucidated, the present study aimed to evaluate the effects of thymus removal on the functional capacity of the immune system after different periods. Methods: A systematic review of the literature was performed using MEDLINE, EMBASE, LILACS and Scopus. The inclusion criteria were original studies that analyzed any component of the immune system in patients with CHD who had undergone thymectomy during cardiac surgery in the first years of life. The results were evaluated for the quality of evidence. Results: Twenty-three studies were selected and showed that patients who underwent a thymectomy in the first years of life tended to exhibit important alterations in the T cell compartment, such as fewer total T cells, CD4+, CD8+, naïve and CD31+ T cells, lower TRECs, decreased diversity of the TCR repertoire and higher peripheral proliferation (increased Ki-67 expression) than controls. However, the numbers of memory T cells and Treg cells differed across the selected studies. Conclusions: Early thymectomy, either partial or complete, may be associated with a reduction in many T cell subpopulations and TCR diversity, and these alterations may persist during long-term follow-up. Alternative solutions should be studied, either in the operative technique with partial preservation of the thymus or through the autograft of fragments of the gland. Systematic Review Registration: Prospero [157188].

Genetic and Antigenic Diversity of Infectious Bronchitis Virus in South America.

Infectious bronchitis virus (IBV) is one of the economically most important diseases affecting the South American poultry industry. The extensive genomic heterogeneity of IBV is a consequence of high mutation rates and recombination events followed by selection. Nucleotide heterogeneity is much higher in the S1 coding region of the relevant spike protein; thus, the S1 sequence is widely used for the IBV genetic classification in genotypes and lineages. Two main lineages (GI-11 and GI-16) extensively circulate in South American chicken flocks. The GI-11 lineage, found exclusively in South America, emerged in the 1950s and is currently the predominant lineage in Brazil and Uruguay. The GI-16 lineage emerged around 1979 and is now circulating in most South American regions. All South American countries include Massachusetts-type strains (GI-1 lineage) in the IBV vaccination programs. The GI-11 and GI-16 lineages display very low antigenic relatedness to Massachusetts vaccine strains. Because these vaccine strains may not confer complete protection against South American lineages, other vaccination strategies have been reported to control GI-11 and GI-16 outbreaks. Analysis of the few full-length genomes of South American strains highlights a complex recombination history of IBV in the continent. A broader geographic and temporal sampling is needed to understand the pattern of genetic variability and the evolutionary history of IBV variants in South America.

Estudio recapitulativo- Diversidad genética y antigénica del virus de la bronquitis infecciosa en América del Sur. El virus de la bronquitis infecciosa es una de las enfermedades económicamente más importantes que afecta a la industria avícola sudamericana. La extensa heterogeneidad genómica de este virus es una consecuencia de las altas tasas de mutación y de los eventos de recombinación seguidos por selección. La heterogeneidad de nucleótidos es mucho mayor en la región codificante S1 de la proteína de la espícula; por tanto, la secuencia S1 se usa ampliamente para la clasificación genética de este virus en genotipos y linajes. Dos linajes principales (GI-11 y GI-16) circulan ampliamente en las parvadas de pollos de América del Sur. El linaje GI-11, que hasta ahora se encuentra exclusivamente en América del Sur, surgió en la década de los aþos 1950s y actualmente es el linaje predominante en Brasil y Uruguay. El linaje GI-16 surgió alrededor de 1979 y ahora está circulando en la mayoría de las regiones de América del Sur. Todos los países de América del Sur incluyen cepas tipo Massachusetts (linaje GI-1) en los programas de vacunación contra la bronquitis infecciosa. Los linajes GI-11 y GI-16 muestran una relación antigénica muy baja con las cepas de la vacuna de Massachusetts. Debido a que estas cepas de la vacuna pueden no conferir una protección completa contra los linajes sudamericanos, se han reportado otras estrategias de vacunación para controlar los brotes de GI-11 y GI-16. El análisis de los pocos genomas completos de cepas sudamericanas destaca una compleja historia de recombinación del virus de la bronquitis en el continente. Se necesita un muestreo geográfico y temporal más amplio para comprender el patrón de variabilidad genética y la historia evolutiva de las variantes del virus de la bronquitis infecciosa en América del Sur.

Avaliação da resposta de anticorpos IgG contra diferentes variantes alélicas do Antígeno 1 de Membrana Apical (AMA-1) de Plasmodium vivax em indivíduos de áreas endêmicas de malária / Evaluation of IgG antibody response against different variants of Plasmodium vivax apical membrane antigen 1 (AMA-1) in individuals from malaria endemic areas

O Plasmodium vivax é a espécie com maior distribuição geográfica no mundo e a que predomina nas Américas, incluindo o Brasil. Comparado ao Plasmodium falciparum, poucas vacinas contra o P. vivax encontram-se em fase de testes clínicos. Um dos antígenos de formas sanguíneas de P. vivax candidato a vacina é o Antígeno 1 de Membrana Apical (PvAMA-1). Entretanto, a diversidade antigênica do mesmo na natureza representa um grande desafio para seu uso no desenvolvimento de uma vacina de ampla cobertura. No presente estudo, avaliamos se os polimorfismos de sequências já descritos são capazes de influenciar na eficácia de uma vacina baseada em PvAMA-1. Para isso, geramos 9 proteínas recombinantes a partir da levedura Pichia pastoris, as quais são representativas de diferentes variantes alélicas do antígeno PvAMA-1, a saber: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS. Após expressão e purificação das proteínas selecionadas, avaliamos comparativamente por ELISA a resposta de anticorpos IgG naturalmente adquiridos em indivíduos expostos a malária, procedentes da Região Amazônica. Todas as proteínas foram obtidas com rendimento e pureza apropriados para os estudos propostos. A prevalência total de indivíduos expostos a malária com anticorpos contra PvAMA-1 Belem foi de 53,68%, em 611 amostras de soro testadas. Entre 100 das amostras sorologicamente positivas para PvAMA-1 Belem, os maiores valores de DO492 foram obtidos para as variantes Chesson I, SK0814 e Sal-1, sugerindo que epítopos comuns ou de reatividade cruzada estão sendo reconhecidos nessas variantes. Por outro lado, níveis mais baixos de DO492 foram obtidos para as variantes Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU e PNG_68_MAS, o que pode significar que essas variantes são menos prevalentes ou não circulam no Brasil. Soros policlonais de camundongos C57BL/6 previamente imunizados com PvAMA-1 Belem foram testados quanto ao reconhecimento das diferentes variantes por ELISA. Nossos resultados demonstraram que as variantes Chesson I, Indonesia XIX, SK0814, Sal-1 e a proteína homóloga foram predominantemente reconhecidas. Por fim, ensaios de competição baseados em ELISA revelaram que as proteínas Chesson I, Indonesia XIX, SK0814 e Sal-1, na fase solúvel, foram capazes de inibir a ligação de anticorpos à variante Belem aderida a placa, sugerindo a presença de epítopos comuns ou de reatividade cruzada entre as mesmas. Nossos dados sugerem que uma vacina baseada na variante PvAMA-1 Belem gera anticorpos variante-transcendentes. Entretanto, para gerar uma vacina universal baseada em PvAMA-1, uma formulação multi-alélica, incluindo variantes da Tailândia e Papua Nova Guiné, deverão ser testadas

Plasmodium vivax has the largest geographical distribution Plasmodium species in the world, and is predominant in the Americas, including Brazil. Fewer P. vivax vaccines than P. falciparum vaccines have successfully reached clinical trials. One of the candidate antigens for a blood-stage P. vivax vaccine is the apical membrane antigen 1 (PvAMA-1). However, the high natural variability found in this antigen presents a major challenge for its development into a wide-range vaccine. In the present study, we evaluated whether sequence polymorphisms would influence a vaccine based on PvAMA-1. To achieve this, we generated 9 recombinant proteins from the yeast Pichia pastoris, representative of different allelic variants of the PvAMA-1 antigen: Belem, Chesson I, Sal-1, Indonesia XIX, SK0814, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS. After expression and purification of these proteins, we compared, by ELISA and IgG blocking, the natural acquired response from malaria-exposed individuals in the Amazon Region. All proteins selected had the appropriate yield and purity for the proposed studies. The total prevalence of malaria-exposed individuals with reactivity to PvAMA-1 Belem was 53,68%, from 611 serum samples tested. One hundred of these serologically positive samples were further tested against recombinant proteins representing the other allelic variants. The highest OD values resulted from Sal-1, Chesson I and SK0814 variants, suggesting that common epitopes or cross-reactivity exist across the variants. On the other hand, the lowest OD values resulted from the variants Indonesia XIX, TC103, PNG_05_ESP, PNG_62_MU, and PNG_68_MAS, which may mean these variants are less prevalent or do not circulate in Brazil. Polyclonal sera from C57BL/6 mice immunized with PvAMA-1 Belem were tested for recognition of different variants by ELISA. Our results showed that the variants Chesson I, Sal-1, Indonesia XIX, SK0814 and the homologous protein were predominantly recognized. Lastly, ELISA-based competition assays revealed that Chesson I, Sal-1, Indonesia XIX and SK0814 proteins were able to inhibit antibody binding to the Belem variant, suggesting the presence of common epitopes or cross-reactivity between these variants. Our data suggest that a vaccine based on the PvAMA-1 Belem variant displays strain-transcendent antibodies. However, to generate a universal vaccine based on PvAMA-1, a multiallelic formulation including variants from Thailand and Papua New Guinea must be tested

About the necessity of including HoBi-like pestiviruses in bovine respiratory and reproductive viral vacines / Sobre a necessidade de incluir pestivírus HoBi-like em vacinas virais respiratórias e reprodutivas para bovinos

HoBi-like pestiviruses (HoBiPeV) constitute a novel group of bovine pestiviruses, genetically and antigenically related to bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Recent data shows that HoBiPeV are endemic among Brazilian cattle, yet bovine reproductive/respiratory vaccines contain only BVDV-1 and BVDV-2 strains. The present study investigated the neutralizing antibody response against these pestiviruses induced by two commercial vaccines (VA = attenuated, VI = inactivated) and by three experimental, replicative, vaccine formulations (VAC1 = monovalent, BVDV-1; VAC2 = bivalent, BVDV-1 + BVDV-2; VAC3 = trivalent, BVDV-1 + BVDV-2 and HoBiPeV). Seronegative beef calves were immunized once (replicative vaccines) or twice (inactivated vaccine) and serum samples were tested by virus-neutralization (VN) 30 days after vaccination (dpv) (replicative vaccines) or 30 days after the second dose (VI). We considered a threshold VN titer of ≥60 indicative of protection against clinical disease. At 30 dpv, VA induced protective titers against BVDV-2 in 7/7 animals (GMT=289.8) and against BVDV-1 and HoBiPeV in 5/7 animals (GMTs=97.5 and 80, respectively). VI induced protective titers against BVDV-1 in 1/7 animal (GMT=16.4), 2/7 animals against BVDV-2 (GMT=53.8) and in none of the calves against HoBiPeV (GMT=12.2). When a pool of sera of each vaccine group was tested against individual Brazilian isolates, VA induced protective titers against 3/7 BVDV-1 isolates, to 9/10 (BVDV-2) and 1/8 (HoBiPeV); VI induced protective titers against 1/7 (BVDV-1), 1/10 (BVDV-2) and none (0/8) HoBiPeV isolates. The experimental vaccine VAC1 induced protective titers against BVDV-1 in 9/9 animals (GMT=320) but in no animal against BVDV-2 or HoBiPeV (GMT<10). VAC2 induced protective titers to BVDV-1 and BVDV-2 in 9/9 animals (GMTs=160 and 640, respectively), and against HoBiPeV in 7/9 animals (GMT=108.5). Finally, VAC3 induced protective titers in all animals against BVDV-1 (GMT=234.3), BVDV-2 (294.9) and HoBiPeV (201.1). Testing the pool of sera against pestivirus isolates, VAC1 induced titers ≥ 60 against 4/7 BVDV-1 but to none BVDV-2/HoBiPeV isolate; VAC2 induced protective titers against 4/7 BVDV-1; 10/10 BVDV-2 and 2/8 HoBiPeV; VAC3 induced protective titers against all BVDV-1, BVDV-2 and HoBiPeV isolates. These results indicate that vaccines composed by BVDV-1+BVDV-2, especially those containing inactivated virus, may not induce serological response against a variety of HoBiPeV isolates. Thus, the need of inclusion of HoBiPeV in vaccine formulations should be considered.(AU)

Os pestivírus HoBi-like (HoBiPeV) compõe um grupo novo de pestivírus de bovinos, genética e antigenicamente relacionados com os vírus da diarreia viral bovina 1 e 2 (BVDV-1, BVDV2). Dados recentes indicam que os HoBiPeV são endêmicos na população bovina do Brasil, mas as vacinas respiratórias e reprodutivas bovinas contêm apenas cepas de BVDV-1 e BVDV-2. O presente estudo investigou a atividade neutralizante contra estes pestivírus induzidas por duas vacinas comerciais (VA = atenuada, VI = inativada) e por três vacinas experimentais replicativas (VAC1 = monovalente, BVDV-1; VAC2 = bivalente, BVDV-1 + BVDV-2; VAC3 = trivalente, BVDV-1 + BVDV-2 e HoBiPeV). Bezerros soronegativos foram imunizados uma vez (vacinas replicativas) ou duas (vacina inativada) e amostras de soro foram testadas por vírus-neutralização (VN) 30 dias após a vacinação (dpv) (vacinas replicativas) ou 30 dias após a segunda dose (VI). Títulos neutralizantes ≥60 foram considerados indicativos de proteção contra doença clínica. Nesta data, a VA induziu títulos protetivos contra o BVDV-2 em 7/7 animais (GMT=289,8) e contra BVDV-1 e HoBiPeV em 5/7 animals (GMTs=97,5 e 80, respectivamente). VI induziu títulos protetores contra BVDV-1 em 1/7 animal (GMT=16,4), em 2/7 animais contra BVDV-2 (GMT=53,8) e em nenhum contra HoBiPeV (GMT=12,2). Quando um pool de soro de cada grupo vacinal foi testado frente a isolados Brasileiros, a VA induziu títulos protetores contra 3/7 isolados de BVDV-1, 9/10 (BVDV-2) e 1/8 (HoBiPeV); VI induziu títulos protetores em 1/7 contra BVDV-1, 1/10 (BVDV-2) e em nenhum (0/8) contra isolados de HoBiPeV. A VAC1 induziu títulos protetores contra BVDV-1 em 9/9 animais (GMT=320) mas em nenhum animal contra BVDV-2 ou HoBiPeV (GMT<10). VAC2 induziu títulos protetores contra BVDV-1e BVDV-2 em 9/9 animais (GMTs=160 e 640, respectivamente),e contra HoBiPeV em 7/9 animais (GMT=108,5). Finalmente, VAC3 induziu títulos protetores em todos os animais contra BVDV-1 (GMT=234,3), BVDV-2 (294,9) e HoBiPeV (201,1). No teste de pool de soro contra isolados de pestivírus, VAC1 induziu títulos ≥60 contra 4/7 BVDV-1 mas contra nenhum isolado de BVDV-2/HoBiPeV; VAC2 induziu títulos protetores contra 4/7 BVDV-1; 10/10 BVDV-2 e 2/8 HoBiPeV; VAC3 induziu títulos protetores contra todos BVDV-1, BVDV-2 e HoBiPeV. Esses resultados indicam que vacinas contendo apenas BVDV-1 BVDV-2, especialmente aquelas inativadas, podem não conferir resposta sorológica protetora contra vários isolados de HoBiPeV. Portanto, a necessidade de se incluir cepas de HoBiPeV nas vacinas deve ser considerada.(AU)

About the necessity of including HoBi-like pestiviruses in bovine respiratory and reproductive viral vacines / Sobre a necessidade de incluir pestivírus HoBi-like em vacinas virais respiratórias e reprodutivas para bovinos

HoBi-like pestiviruses (HoBiPeV) constitute a novel group of bovine pestiviruses, genetically and antigenically related to bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2. Recent data shows that HoBiPeV are endemic among Brazilian cattle, yet bovine reproductive/respiratory vaccines contain only BVDV-1 and BVDV-2 strains. The present study investigated the neutralizing antibody response against these pestiviruses induced by two commercial vaccines (VA = attenuated, VI = inactivated) and by three experimental, replicative, vaccine formulations (VAC1 = monovalent, BVDV-1; VAC2 = bivalent, BVDV-1 + BVDV-2; VAC3 = trivalent, BVDV-1 + BVDV-2 and HoBiPeV). Seronegative beef calves were immunized once (replicative vaccines) or twice (inactivated vaccine) and serum samples were tested by virus-neutralization (VN) 30 days after vaccination (dpv) (replicative vaccines) or 30 days after the second dose (VI). We considered a threshold VN titer of ≥60 indicative of protection against clinical disease. At 30 dpv, VA induced protective titers against BVDV-2 in 7/7 animals (GMT=289.8) and against BVDV-1 and HoBiPeV in 5/7 animals (GMTs=97.5 and 80, respectively). VI induced protective titers against BVDV-1 in 1/7 animal (GMT=16.4), 2/7 animals against BVDV-2 (GMT=53.8) and in none of the calves against HoBiPeV (GMT=12.2). When a pool of sera of each vaccine group was tested against individual Brazilian isolates, VA induced protective titers against 3/7 BVDV-1 isolates, to 9/10 (BVDV-2) and 1/8 (HoBiPeV); VI induced protective titers against 1/7 (BVDV-1), 1/10 (BVDV-2) and none (0/8) HoBiPeV isolates. The experimental vaccine VAC1 induced protective titers against BVDV-1 in 9/9 animals (GMT=320) but in no animal against BVDV-2 or HoBiPeV (GMT<10). VAC2 induced protective titers to BVDV-1 and BVDV-2 in 9/9 animals (GMTs=160 and 640, respectively), and against HoBiPeV in 7/9 animals (GMT=108.5). Finally, VAC3 induced protective titers in all animals against BVDV-1 (GMT=234.3), BVDV-2 (294.9) and HoBiPeV (201.1). Testing the pool of sera against pestivirus isolates, VAC1 induced titers ≥ 60 against 4/7 BVDV-1 but to none BVDV-2/HoBiPeV isolate; VAC2 induced protective titers against 4/7 BVDV-1; 10/10 BVDV-2 and 2/8 HoBiPeV; VAC3 induced protective titers against all BVDV-1, BVDV-2 and HoBiPeV isolates. These results indicate that vaccines composed by BVDV-1+BVDV-2, especially those containing inactivated virus, may not induce serological response against a variety of HoBiPeV isolates. Thus, the need of inclusion of HoBiPeV in vaccine formulations should be considered.(AU)

Os pestivírus HoBi-like (HoBiPeV) compõe um grupo novo de pestivírus de bovinos, genética e antigenicamente relacionados com os vírus da diarreia viral bovina 1 e 2 (BVDV-1, BVDV2). Dados recentes indicam que os HoBiPeV são endêmicos na população bovina do Brasil, mas as vacinas respiratórias e reprodutivas bovinas contêm apenas cepas de BVDV-1 e BVDV-2. O presente estudo investigou a atividade neutralizante contra estes pestivírus induzidas por duas vacinas comerciais (VA = atenuada, VI = inativada) e por três vacinas experimentais replicativas (VAC1 = monovalente, BVDV-1; VAC2 = bivalente, BVDV-1 + BVDV-2; VAC3 = trivalente, BVDV-1 + BVDV-2 e HoBiPeV). Bezerros soronegativos foram imunizados uma vez (vacinas replicativas) ou duas (vacina inativada) e amostras de soro foram testadas por vírus-neutralização (VN) 30 dias após a vacinação (dpv) (vacinas replicativas) ou 30 dias após a segunda dose (VI). Títulos neutralizantes ≥60 foram considerados indicativos de proteção contra doença clínica. Nesta data, a VA induziu títulos protetivos contra o BVDV-2 em 7/7 animais (GMT=289,8) e contra BVDV-1 e HoBiPeV em 5/7 animals (GMTs=97,5 e 80, respectivamente). VI induziu títulos protetores contra BVDV-1 em 1/7 animal (GMT=16,4), em 2/7 animais contra BVDV-2 (GMT=53,8) e em nenhum contra HoBiPeV (GMT=12,2). Quando um pool de soro de cada grupo vacinal foi testado frente a isolados Brasileiros, a VA induziu títulos protetores contra 3/7 isolados de BVDV-1, 9/10 (BVDV-2) e 1/8 (HoBiPeV); VI induziu títulos protetores em 1/7 contra BVDV-1, 1/10 (BVDV-2) e em nenhum (0/8) contra isolados de HoBiPeV. A VAC1 induziu títulos protetores contra BVDV-1 em 9/9 animais (GMT=320) mas em nenhum animal contra BVDV-2 ou HoBiPeV (GMT<10). VAC2 induziu títulos protetores contra BVDV-1e BVDV-2 em 9/9 animais (GMTs=160 e 640, respectivamente),e contra HoBiPeV em 7/9 animais (GMT=108,5). Finalmente, VAC3 induziu títulos protetores em todos os animais contra BVDV-1 (GMT=234,3), BVDV-2 (294,9) e HoBiPeV (201,1). No teste de pool de soro contra isolados de pestivírus, VAC1 induziu títulos ≥60 contra 4/7 BVDV-1 mas contra nenhum isolado de BVDV-2/HoBiPeV; VAC2 induziu títulos protetores contra 4/7 BVDV-1; 10/10 BVDV-2 e 2/8 HoBiPeV; VAC3 induziu títulos protetores contra todos BVDV-1, BVDV-2 e HoBiPeV. Esses resultados indicam que vacinas contendo apenas BVDV-1 BVDV-2, especialmente aquelas inativadas, podem não conferir resposta sorológica protetora contra vários isolados de HoBiPeV. Portanto, a necessidade de se incluir cepas de HoBiPeV nas vacinas deve ser considerada.(AU)

Phylogenetic, Evolutionary and Structural Analysis of Canine Parvovirus (CPV-2) Antigenic Variants Circulating in Colombia.

Canine parvovirus (CPV-2) is the causative agent of haemorrhagic gastroenteritis in canids. Three antigenic variants-CPV-2a, CPV-2b and CPV-2c-have been described, which are determined by variations at residue 426 of the VP2 capsid protein. In Colombia, the CPV-2a and CPV-2b antigenic variants have previously been reported through partial VP2 sequencing. Mutations at residues Asn428Asp and Ala514Ser of variant CPV-2a were detected, implying the appearance of a possible new CPV-2a variant in Colombia. The purpose of the present study was to characterise the full VP2 capsid protein in samples from Antioquia, Colombia. We conducted a cross-sectional study with 56 stool samples from dogs showing clinical symptoms of parvoviral disease. Following DNA extraction from the samples, VP2 amplification was performed using PCR and positive samples were sequenced. Sequence and phylogenetic analyses were performed by comparison with the VP2 gene sequences of the different CPV-2 worldwide. VP2 was amplified in 51.8% of the analysed samples. Sequencing and sequence alignment showed that 93.1% of the amplified samples belonged to the new CPV-2a antigenic variant previously. Analysing the amino acid sequences revealed that all CPV-2a contain Ala297Asn mutations, which are related to the South America I clade, and the Ala514Ser mutation, which allows characterization as a new CPV-2a sub-variant. The Colombian CPV-2b variant presented Phe267Tyr, Tyr324Ile and Thr440Ala, which are related to the Asia-I clade variants. The CPV-2c was not detected in the samples. In conclusion, two antigenic CPV-2 variants of two geographically distant origins are circulating in Colombia. It is crucial to continue characterising CPV-2 to elucidate the molecular dynamics of the virus and to detect new CPV-2 variants that could be becoming highly prevalent in the region.

Antigenic characterization of novel H1 influenza A viruses in swine.

Novel H1N2 influenza A viruses (IAVs) in swine have been identified in Chile co-circulating with pandemic H1N1 2009-like (A(H1N1)pdm09-like) viruses. The objective of this study was to characterize antigenically the swine H1 IAVs circulating in Chile. Genetic analysis based on the HA1 domain and antigenic analysis by hemagglutination inhibition assay were carried out. Three antigenic clusters were identified, named Chilean H1 A (ChH1A), Chilean H1 B (ChH1B), and A(H1N1)pdm09-like. The antigenic sites of ChH1A and ChH1B strains were 10-60% distant from those of commercial vaccine strains at the amino acid sequence level. Antigenic variants were identified within the clusters ChH1A and A(H1N1)pdm09-like. Substitutions in the main antigenic sites (E153G in Sa, Q193H in Sb, D168N in Ca1, P137S in Ca2, and F71L in Cb) were detected in variants from the ChH1A cluster, whereas only a single substitution in antigenic site Sa (G155E) was detected in variants from A(H1N1)pdm09-like cluster, which confirms the importance to carrying out antigenic analyses in addition to genetic analyses to evaluate control measures such as vaccination. These results highlight the need to update vaccines for swine in Chile and the importance of continued surveillance to determine the onward transmission of antigenic variants in Chilean pig populations.

SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

Phylodynamic analyses of Brazilian antigenic variants of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a very important pathogen to poultry production and it is classified into three main groups: classical virulent (cvIBDV), very virulent (vvIBDV) and antigenic variants (avIBDV). This last group is composed by five different genetic lineages (recently classified in genogroups G2, G4, G5, G6, and G7) distributed in specific regions around the world. Brazil is one of the biggest poultry producers in the world and the present study aimed to investigate the evolutionary history of avIBDVs of the genogroup G4 in Brazil. A total of 5331 IBDV positive bursa samples, from different Brazilian poultry flocks, were genotyped in a period of ten years (2005 to 2014) and 1888 (35.42%) were identified as local avIBDVs. The highly variable region of the viral protein 2 (hvvp2) gene of 28 avIBDVs was sequenced and used in phylogenetic analyses and evaluation of local amino acid signatures. In addition, all complete and partial IBDV vp2 gene sequences, with local and year of collection information available on GenBank, were retrieved. Phylogenetic analyses were carried out based on a maximum likelihood method for the classification of genogroups occurring in Brazil. Based on a Maximum Likelihood (ML) phylogenetic tree, all Brazilian avIBDVs grouped into the genogroup 4. Bayesian phylodynamics analysis demonstrated the ancestor virus of this group was probably introduced in South America in 1968 (1960 to 1974, 95% HPD) and in Brazil in 1974 (1968 to 1977, 95% HPD) and the most likely source was East Europe (Hungary or Poland). All Brazilian avIBDV sequences, as well as the other genogroup 4 sequences, showed a specific pattern of amino acid: S222, T272, P289, I290, and F296. This report brings new insights about the IBDV epidemiology in Brazil and South America.

Genetic and antigenic heterogeneity of infectious bronchitis virus in South America: implications for control programmes.

Infectious bronchitis virus (IBV) is a persistent sanitary problem for the South American poultry industry despite extensive vaccination. The IBV single-stranded RNA genome has high rates of mutation and recombination that generate a notorious virus variability. Since most IBV vaccines are type-specific, there is a need for constant surveillance of the circulating lineages and knowledge about their genetic and antigenic properties. Here we present an integrative analysis that provides the pattern of genetic variation of the South American IBV strains and information about their antigenic characteristics. The genetic analysis was performed using the S1 complete coding sequences of all available South American strains, including newly obtained Argentine and Uruguayan field samples. Our phylogenetic and phylodynamic analyses evidence that three main lineages (GI-1, GI-11 and GI-16) are extensively circulating in South American flocks. Strains of the GI-1 lineage (Massachusetts-type) were detected in Argentina, Brazil, Chile and Colombia. The GI-11 lineage is an exclusively South American lineage that emerged in the 1950s, and is the predominant lineage in Brazil and Uruguay at present. The GI-16 lineage emerged around 1979, and is currently circulating in most South American territories (Argentina, Chile, Uruguay, Colombia and Peru). The virus cross-neutralization test performed here reveals very low antigenic relatedness between GI-11 and GI-16 lineages (i.e. they are different serotypes). The results of this study extend our knowledge about the present and past IBV variability in South America and provide relevant elements to improve the control programmes by considering the genetic and antigenic attributes of IBV.

Evaluation of mechanisms that may generate DNA lesions triggering antigenic variation in African trypanosomes.

Antigenic variation by variant surface glycoprotein (VSG) coat switching in African trypanosomes is one of the most elaborate immune evasion strategies found among pathogens. Changes in the identity of the transcribed VSG gene, which is always flanked by 70-bp and telomeric repeats, can be achieved either by transcriptional or DNA recombination mechanisms. The major route of VSG switching is DNA recombination, which occurs in the bloodstream VSG expression site (ES), a multigenic site transcribed by RNA polymerase I. Recombinogenic VSG switching is frequently catalyzed by homologous recombination (HR), a reaction normally triggered by DNA breaks. However, a clear understanding of how such breaks arise-including whether there is a dedicated and ES-focused mechanism-is lacking. Here, we synthesize data emerging from recent studies that have proposed a range of mechanisms that could generate these breaks: action of a nuclease or nucleases; repetitive DNA, most notably the 70-bp repeats, providing an intra-ES source of instability; DNA breaks derived from the VSG-adjacent telomere; DNA breaks arising from high transcription levels at the active ES; and DNA lesions arising from replication-transcription conflicts in the ES. We discuss the evidence that underpins these switch-initiation models and consider what features and mechanisms might be shared or might allow the models to be tested further. Evaluation of all these models highlights that we still have much to learn about the earliest acting step in VSG switching, which may have the greatest potential for therapeutic intervention in order to undermine the key reaction used by trypanosomes for their survival and propagation in the mammalian host.

Avaliação da imunogenicidade de diferentes formas alélicas da proteí­na recombinante PvAMA-1expressa em Pichia pastoris: impacto da diversidade antigênica / Evaluation of the immunogenicity of different allelic forms of PvAMA-1 protein expressed in Pichia pastoris: impact of antigenic diversity

A malária é um problema de saúde pública no Brasil e no mundo. Em 2016, o número de casos estimado pela Organização Mundial de Saúde foi de 216 milhões. Plasmodium falciparum é a espécie mais prevalente e responsável pelo maior número de mortes no mundo, sobretudo no continente africano. Por outro lado, o Plasmodium vivax é conhecido por sua ampla distribuição geográfica, sendo a espécie que predomina nas Américas, incluindo o Brasil. Nos últimos 20 anos, nosso grupo tem gerado e caracterizado diversas proteínas recombinantes baseadas em antígenos imunodominantes de P. vivax que podem servir como base para o desenvolvimento de uma vacina contra malária. Entre os antígenos de merozoítas, uma das principais proteínas em estudo pelo nosso grupo é o Antígeno 1 de Membrana Apical de P. vivax (PvAMA-1), caracterizado previamente como altamente imunogênico em infecções naturais e em camundongos imunizados, na presença de diferentes adjuvantes. O objetivo do presente estudo foi investigar o efeito da diversidade antigênica dessa proteína no reconhecimento por anticorpos específicos e na indução de imunidade contra o parasita. Para isso, foram geradas seis novas proteínas representando diferentes alelos descritos na natureza: PvAMA-1-Belem, PvAMA-1-Sal-I, PvAMA-1-Chesson-I, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX e PvAMA-1-PNG_62_MU. As proteínas recombinantes foram expressas em leveduras Pichia pastoris e purificadas em duas etapas cromatográficas. Em seguida, as imunizações em camundongos C57BL/6 foram realizadas com as proteínas administradas de forma isolada, ou em combinação, na presença do adjuvante agonista de TLR3 (Poly I:C). Por ELISA, observamos que todas as formulações foram capazes de induzir anticorpos IgG contra as proteínas homólogas e heterólogas, o que sugere que a diversidade antigênica entre as formas alélicas não compromete o reconhecimento. Os dados gerados no presente trabalho sugerem que uma formulação contendo mistura de diferentes alelos representando a proteína AMA-1 pode ser explorada para o desenvolvimento de uma vacina de ampla cobertura contra o P. vivax

Malaria is a public health problem in Brazil and throughout the world. In 2016, the World Health Organization estimated there were 216 million cases of malaria. Plasmodium falciparum is the most prevalent species and is responsible for the largest number of deaths, especially in the African continent. However, Plasmodium vivax is known for its wide geographic distribution, being the species that prevails in the Americas, including Brazil. In the last 20 years, our group has generated and characterized several recombinant proteins based on immunodominant antigens of P. vivax that can serve as a basis for the development of a malaria vaccine. Among the merozoite antigens, one of the main proteins studied by our group is P. vivax apical membrane antigen-1 (PvAMA-1), previously characterized as highly immunogenic in natural infections and immunized mice, in the presence of different adjuvants. The objective of this study was to investigate the effect of antigenic diversity of this protein in the recognition of specific antibodies and the induction of immunity against the parasite. For this, six new proteins were generated representing different alleles described in nature: PvAMA-1-Belem, PvAMA-1-Sal-i, PvAMA-1-Chesson-i, PvAMA-1-SK0814-apical, PvAMA-1-Indonesia-XIX, and PvAMA-1-PNG_62_MU. Recombinant proteins were expressed in Pichia pastoris yeast and purified by two chromatographic stages. Then, C57BL/6 mice were immunized with these proteins administered in isolation or in combination, in the presence of the TLR3 agonist adjuvant, Poly I:C. Using an enzyme-linked immunosorbent assay, we observed that all formulations induced IgG antibodies against homologous and heterologous proteins. This indicates that antigenic diversity between allele forms does not compromise recognition. This finding suggests that a formulation containing a mixture of different alleles representing the PvAMA-1 protein can be exploited for developing of a wide coverage vaccine against P. vivax

Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses.

Hobi-like viruses comprise an unclassified group of bovine pestiviruses related to bovine viral diarrhea virus 1 (BVDV-1) and 2 (BVDV-2). These viruses were originally identified in fetal bovine serum from Brazilian origin and, subsequently, isolated from diseased animals in several countries. Herein we performed an antigenic characterization of eight Brazilian HoBi-like viruses isolated from persistently infected (PI) animals and from gastroenteric disease (2007-2015). Phylogenetic analysis based on the 5' unstranslated region (UTR) clustered these viruses with other HoBi-like viruses from European and Asiatic origin. Monoclonal antibody (MAb) binding indicated variability in the Hobi-like virus glycoprotein E2 and significant differences from the homologous BVDV-1 and BVDV-2 glycoprotein. Analysis of antigenic relatedness based on virus-neutralizing titers using virus-specific antisera revealed that HoBi-like viruses are antigenically very different from BVDV-1 and, to a lesser extent, from BVDV-2. Cross-neutralizing assays between pairs of HoBi-like viruses and their respective antisera indicated the existence of antigenic variability among these viruses, even for viruses isolated from the same herd in different occasions. Moreover, the identification of a HoBi-like isolate with low antigenic similarity with the other isolates indicates the potential existence of antigenic subgroups among HoBi-like virus isolates. Finally, sera of lambs immunized with commercial BVDV vaccines showed low or undetectable neutralizing activity against HoBi-like isolates. These results indicate significant antigenic differences between BVDV genotypes and Brazilian HoBi-like viruses and the existence of antigenic variability within this atypical group of pestiviruses. These findings extend the knowledge about the antigenic diversity of HoBi-like viruses and reinforce the need for their inclusion in current BVDV vaccines.