In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

Bees are fundamental in several aspects, especially in relation to plant biodiversity and pollination. Recently, immense losses are being faced in the number of Brazilian colonies, mainly in southern states of the country, which has a strong beekeeping activity. There are indications that, among the reasons for the losses, pathogens that affect the health of bees may be involved. Among them, the microsporidium Nosema and the black queen cell virus (BQCV) stand out for their prevalence. In this study, 92 colonies of 17 apiaries from southern Brazil were evaluated for infection by Nosema ceranae, Nosema apis and BQCV. Nucleic acid extractions and cDNA synthesis were performed from adult bee samples, followed by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and multiplex PCR. Eight BQCV positive samples were subjected to sequencing. The results showed that N. ceranae and BQCV are circulating in the Southern region of the country, which may be the reason for the loss of colonies. N. apis was not found. N. ceranae was found in 57.6% (53/92) of the colonies and BQCV in 32.6% (30/92). Co-infection was found in 25% (23/92) of the colonies studied, a factor that is suggested to be reducing the hosts’ longevity due to the synergistic action of the pathogens. The samples submitted to sequencing indicated similarity of 96.8 to 100% between them, in addition to strong similarity with sequences from Asia, United States, Germany and Peru. This study reports the circulation of N. ceranae and BQCV in apiaries in southern Brazil, in addition to being the first phylogenetic analysis of the Brazilian BQCV sequence.

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O sistema imune presente nas mucosas representa a barreira inicial frente à infecção por diversos patógenos que utilizam estas superfícies como porta de entrada no organismo hospedeiro, como é o caso dos Alphaherpesvirus bovino 1 (BoHV-1) e Alphaherpesvirus bovino 5 (BoHV-5). Estes vírus infectam os sistemas reprodutivo e respiratório de bovinos e são responsáveis por casos de Rinotraqueíte Infecciosa Bovina, Vulvovaginite Pustular Infecciosa e Balanopostite Pustular Infecciosa, assim como Meningoencefalite Herpética Bovina, respectivamente. A infecção pelos BoHVs ocorre nas cavidades revestidas por células epiteliais de mucosa, principalmente nasal e genital, sendo o ponto inicial de replicação, seguindo-se a disseminação local, viremia eventual e disseminação neuronal, até o estabelecimento da latência. A cooperação dos mecanismos de defesa confere proteção através do sistema integrado de mucosas por meio da ativação dos sítios efetores e especialmente produção de diferentes isotipos de imunoglobulinas, como a SIgA, realiza neutralização nas superfícies mucosas, e a IgG, que desempenha neutralização na mucosa e sistemicamente. A indução da imunidade nas superfícies mucosas através do uso de vacinas torna-se de extrema importância para a prevenção da adsorção do vírus à célula e infecção pelos microrganismos patogênicos e depende, especialmente, do imunógeno, via de imunização e substância adjuvante empregada nas formulações. Esta revisão teve como objetivo abordar os principais aspectos relacionados à infecção pelo BoHV-1 e BoHV-5, assim como os tipos de resposta relacionadas ao sistema imune de mucosas frente a estes vírus e, ainda, estudos relacionados à utilização das vias mucosas para administração de vacinas em diferentes espécies animais.

The search for natural resources with antiviral potential, as an alternative to synthetic drugs, has been growing and, in this sense, oregano presents itself as a potential candidate. However, the antiviral studies with oregano are still poorly explored. BoHV-1 stands out among veterinary pathogens, for its economic impact on cattle production. In this study, the antiviral and virucidal activity of polar extracts of Origanum vulgare was evaluated against BoHV-1. Infusion (INF10), decoction (DEC), and hydroalcoholic (HAE) extracts were tested to cytotoxic and antiviral assays on MDBK cells. Cytotoxic effects were analyzed through MTT assay and the antiviral activity was expressed as a percentage of inhibition (PI). BoHV-1 was incubated with O. vulgare extracts as virucidal assay. Concentrations ≤3.12 mg/ml (INF10) and ≤1.56 mg/ml (DEC/HAE) preserved the cell viability above 60%, and all extracts were safe (>96%) between 0.78 and 0.39 mg/ml. Regarding the antiviral activity, pre-treatment of all extracts highlighted in comparison to the post-treatment. The pre-treatment of infusion at 2 mg/ml highlighted due to the high cell viability (84.69%) and the elimination of the viral load. All extracts inactivated BoHV-1 from 2 hours of incubation (20 mg/ml), showing virucidal activity. These findings may be related to 4-hydroxybenzoic acid as prevalent in all extracts. These findings showed the in vitro antiviral and virucidal activity of oregano polar extracts against BoHV-1 and may be promising for the therapeutic use against herpesviruses infections.