Influenza A virus (IAV) is a single-stranded, negative-sense RNA virus and a common cause of seasonal flu in humans. Its genome comprises eight RNA segments that facilitate reassortment, resulting in a great variety of IAV strains. To study these processes, the genetic code of each segment should be unraveled. Fortunately, new third-generation sequencing approaches allow for cost-efficient sequencing of IAV segments. Sequencing success depends on various factors, including proper sample storage and processing. Hence, this work focused on the effect of storage of oral fluids and swIAV sequencing. Oral fluids (n = 13) from 2017 were stored at −22 °C and later transferred to −80 °C. Other samples (n = 21) were immediately stored at −80 °C. A reverse transcription quantitative PCR (RT-qPCR) pre- and post-storage was conducted to assess IAV viral loads. Next, samples were subjected to two IAV long-read nanopore sequencing methods to evaluate success in this complex matrix. A significant storage-associated loss of swIAV loads was observed. Still, a total of 17 complete and 6 near-complete Polish swIAV genomes were obtained. Genotype T, (H1avN2, seven herds), P (H1N1pdm09, two herds), U (H1avN1, three herds), and A (H1avN1, 1 herd) were circulated on Polish farms. In conclusion, oral fluids can be used for long-read swIAV sequencing when considering appropriate storage and segment amplification protocols, which allows us to monitor swIAV in an animal-friendly and cost-efficient manner.

The 2019 coronavirus disease pandemic (COVID-19) showed how genetic mutations can alter coronavirus characteristics. However, the evolution of livestock coronaviruses remains understudied. We analyzed 15 bovine coronavirus (BCoV), three porcine hemagglutinating encephalomyelitis virus (PHEV) and 18 porcine respiratory coronavirus (PRCV) isolates, mainly from Belgian livestock collected between 2020 and 2023. Spike gene phylogenetic analysis showed nucleotide substitution rates comparable between BCoV and PRCV, while PHEV appeared slower. Unlike severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), synonymous substitutions were preferred, limiting amino acid variation across decades in the animal coronaviruses. Virus neutralization assays with swine antisera indicated minimal antigenic change in PHEV and PRCV. Recent BCoV isolates showed antigenic divergence from the classical Mebus vaccine strain. The impact of this divergence on vaccine efficacy may warrant further research. Our findings underscore the need for periodic surveillance, as changes in surface proteins may affect pathogenicity, tissue tropism, host range and vaccine efficacy.

Mycoplasmas are known respiratory pathogens in tortoises, but few studies exist in snakes. To better understand the correlation with clinical signs and co-infections, samples from mycoplasma-positive snakes with and without clinical respiratory disease were analyzed. Oral swabs from 15 snakes (pythons n = 12, boas n = 3) were examined using polymerase chain reaction (PCR) and third-generation sequencing (TGS). Additionally, mycoplasma isolation assays were performed. Pathogens detected by PCR included Mycoplasmas (15/15, 100%), serpentoviruses (9/15, 60%), and Chlamydia sp. (2/15, 13%); those detected by TGS included Mycoplasmas (14/15, 93%), serpentoviruses (10/15, 67%), Chlamydia sp. (1/15, 7%), and 15 different bacterial species. Sequencing of the mycoplasma PCR products revealed a close genetic relationship to Mycoplasmopsis agassizii. TGS identified genetically distinct mycoplasmas and three different serpentoviruses. While mycoplasmas could not be successfully propagated, Brucella intermedia comb. nov. was identified in eight cultures. Respiratory disease in snakes is often multifactorial, involving various pathogens and environmental influences. This study demonstrates that comprehensive diagnostics are essential for understanding disease processes in snakes and improving the detection of diverse pathogens. Further research is needed to improve laboratory diagnostics for infectious diseases in reptiles and to better understand the roles of various pathogens in respiratory diseases in snakes.