Biology subjects

de Oliveira, P. S. B.

Publications and source records attributed to de Oliveira, P. S. B..

4 recordsLinked to original sources

bioRxiv2026

Hematogenous neuroinvasion and genotype-dependent transmission of influenza A H5N1 viruses in the cat host

The spillover of highly pathogenic avian influenza (HPAI) A H5N1 virus to mammalian hosts raises major concerns due to its pandemic potential. Cats are frequently affected mammals, often succumbing to systemic and neurological disease. Here, we characterized the pathogenesis and transmissibility of two H5N1 genotypes, B3.13 and D1.1, in cats. Infected cats exhibited high-level viremia and virus shedding in nasal, oral, and fecal secretions were consistently detected. The virus replicated initially in the upper respiratory tract and lungs, followed by systemic dissemination and neuroinvasion. Notably, the virus crossed the blood-brain-barrier by infecting endothelial cells, spreading to astrocytes and neurons, causing multifocal encephalitis. D1.1-virus infection caused protracted disease with lower shedding and no transmissibility, whereas B3.13 virus caused rapid onset with efficient shedding and transmission. These findings reveal critical H5N1 neuropathogenesis mechanisms and highlight mammalian transmission potential in a species with close human contact.

microbiology↗

bioRxiv2026

Recombinant influenza A H5N1 viruses expressing fluorescent and luminescent reporter proteins

Highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b viruses present a broad host range, with recent spillover and sustained transmission in dairy cattle reported in the United States. Replication-competent reporter viruses are critical tools that enable real-time monitoring of virus replication facilitating high-throughput screens. In this study, we engineered three recombinant H5N1 clade 2.3.4.4b reporter viruses expressing nanoluciferase (NLuc) and two fluorescent reporter proteins, miniGFP2 and UnaG within the open reading frame of the nonstructural (NS) gene of the bovine A/Cattle/Texas/063224-24-1/2024 (TX2/24) virus. All reporter viruses replicated efficiently in vitro, presenting replication kinetics comparable to the parental rTX2/24 virus, but exhibited smaller plaque sizes suggesting reduced cell-to-cell spread. In vivo infection studies in mice showed comparable pathogenicity among all four viruses, although rTX2/24-miniGFP2 and rTX2/24-UnaG exhibited decreased virus shedding relative to rTX2/24 and rTX2/24-NLuc. Virus titrations and in situ localization of virus replication sites demonstrated robust replication in respiratory tissues, with slightly attenuated systemic dissemination of all three reporter viruses. Fluorescent virus neutralization assays using miniGFP2 and UnaG reporter viruses accurately quantified neutralizing antibody titers in sera from naturally infected dairy cattle, consistent with wild-type virus assays. Additionally, the utility of the NLuc reporter virus for antiviral screening was validated against Oseltamivir in vitro. Collectively, these results establish the TX2/24-based reporter viruses as versatile and biologically relevant tools for investigating H5N1 pathogenesis to be used in serological and antiviral drug screens against H5N1 viruses.

microbiology↗

bioRxiv2026

Infection and transmission dynamics of bovine and human influenza A H5N1 viruses in mouse and hamster models

Here we investigated the pathogenesis and contact transmission of bovine- and human-derived highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b genotype B3.13 viruses in mammalian models. Using reverse genetics, we rescued three naturally occurring viruses: rTX2/24 (bovine-derived), rTexas/37 and rMichigan/90 (both human-derived), and compared their infection dynamics, replication and pathogenicity with the wild-type bovine TX2/24 strain in vitro and in vivo. All four viruses demonstrated comparable replication kinetics in four mammalian cell lines. However, the rMichigan/90 strain exhibited significantly smaller plaques in bovine and human cells. In vivo studies showed that mice infected with any of the viruses succumbed to infection within 4-5 days; however, mice infected with the rMichigan/90 virus exhibited slightly lower viral replication and shedding compared to the other strains. Similarly, as in the mouse experiments, in hamsters, all viruses induced body weight loss and oral shedding, with robust virus replication observed in tissues, but the rMichigan/90 virus presented reduced replication and shedding. Contact transmission studies in hamsters revealed limited transmissibility for these viruses, with only one out of four animals inoculated with the rMichigan/90 virus transmitting it to a naive contact. These findings indicate that both bovine- and human-derived H5N1 genotype B3.13 viruses present high pathogenicity in mammals, though the overall transmissibility remains low. Significance StatementInfluenza A H5N1 virus spilled over from wild birds into dairy cattle in the US in 2024. Since then, an increased number of human infections - with at least 71 confirmed cases - were confirmed. In the present study we evaluated and compared the pathogenicity and transmissibility of a bovine and two human H5N1 viruses of the genotype B3.13. Our results show that while all viruses are pathogenic in mouse and hamster models, the bovine isolate TX2/24 presents a broader tissue tropism than the human derived viruses. Contact transmission studies revealed a limited ability of these viruses to transmit in the hamster contact transmission model. These findings highlight the high pathogenicity of H5N1 viruses in mammals and demonstrate that that their transmissibility potential remains low.

microbiology↗

bioRxiv2025

Stability of influenza A H5N1 virus in raw milk cheese

Here we evaluated the stability of highly pathogenic avian influenza (HPAI) H5N1 virus in raw milk cheeses using a mini cheese model prepared with HPAI-spiked raw milk under varying pH levels (pH 6.6, 5.8 and 5.0) and in commercial raw milk cheese inadvertently produced with naturally contaminated raw milk. We observed a pH-dependent survival of the virus, with infectious virus persisting throughout the cheese making process and for up to 60 days of aging in the pH 6.6 and 5.8 cheese groups. Whereas at pH 5.0, the virus did not survive the cheese making process. These findings were validated using the commercial raw milk cheese samples in which infectious virus was detected for up to 60 days of aging. Our study highlights the potential public health risks of consuming raw milk cheese, underscoring the need for additional mitigation steps in cheese production to prevent human exposure to infectious virus.

microbiology↗