†These authors contributed equally to this publication.Dengue virus nonstructural protein 5 (NS5) is a large multifunctional protein with a central role in viral replication. We previously identified two nuclear localization sequences (NLSs) within the central region of dengue virus type-2 (DENV-2) NS5 ('aNLS' and 'bNLS') that are recognized by the importin a/b and importin b1 nuclear transporters, respectively. Here, we demonstrate the importance of the kinetics of NS5 nuclear localization to virus production for the first time and show that the aNLS is responsible. Site-specific mutations in the bipartitetype aNLS or bNLS region were introduced into a reporter plasmid encoding green fluorescent protein fused to the N-terminus of DENV-2 NS5, as well as into DENV-2 genomic length complementary DNA. Mutation of basic residues in the highly conserved region of the bNLS did not affect nuclear import of NS5. In contrast, mutations in either basic cluster of the aNLS decreased NS5 nuclear accumulation and reduced virus production, with the greatest reduction observed for mutation of the second cluster (K 387 K 388 K 389 ); mutagenesis of both clusters abolished NS5 nuclear import and DENV-2 virus production completely. The latter appeared to relate to the impaired ability of virus lacking nuclear-localizing NS5, as compared with wild-type virus expressing nuclear-localizing NS5, to reduce interleukin-8 production as part of the antiviral response. The results overall indicate that NS5 nuclear localization through the aNLS is integral to viral infection, with significant implications for other flaviviruses of medical importance, such as yellow fever and West Nile viruses.

Studies on human ovarian xenografts and mouse allografts indicate that the male hormonal milieu and exogenous gonadotrophin administration stimulate antral follicle growth. However, it is not known whether oocytes produced under these conditions are developmentally competent. The objective of our study was to evaluate the developmental competence of oocytes produced in heterotopic mouse ovarian grafts placed in male and female recipient mice. Gonadotrophins were 7.5 IU pregnant mare serum gonadotrophin (PMSG) alone or 7.5 IU PMSG and 7.5 IU human chorionic gonadotrophin or were not given prior to oocyte collection. The developmental competence of oocytes was assessed by performing in vitro fertilisation and embryo transfer to recipients. When no gonadotrophins were given the cleavage rate was similar for oocytes collected from ovarian grafts in male and female recipients. Gonadotrophin treatment significantly (P < 0.05) increased two-cell formation by oocytes grown in female graft recipients but not in male recipients. Implantation rates, fetal development and the birth of live young were unaffected by the sex of the graft recipient or gonadotrophin treatment. Live offspring were produced from oocytes collected from ovarian grafts in male and female recipients treated with or without gonadotrophins. In conclusion, this work has shown that the hormonal environment of male mice can support the growth of oocytes in ovarian allografts and that these oocytes can produce live offspring.

Objectives The emergence of the 2019 novel coronavirus (SARS‐CoV‐2) has necessitated evaluation of the potential for SARS‐CoV‐2 infection in dogs and cats. Using a large data set, we evaluated the frequency of SARS‐CoV‐2 and other respiratory pathogens in samples submitted for respiratory testing from mid‐February to mid‐April 2020. Materials and Methods A SARS‐CoV‐2 real‐time PCR was developed and validated. A subset of canine and feline samples submitted for respiratory pathogen panel testing to reference laboratories in Asia, Europe, and North America were also tested for SARS‐CoV‐2. The frequency of respiratory pathogens was compared for the February–April period of 2020 and 2019. Results Samples from 4616 patients were included in the study and 44% of canine and 69% of feline samples were PCR positive with Mycoplasma cynos and Bordetella bronchiseptica and Mycoplasma felis and feline calicivirus, respectively. No SARS‐CoV‐2 infections were identified. Positive results for respiratory samples were similar between years. Clinical Significance The data in this study suggest that during the emergence of the SARS‐CoV‐2 pandemic in early 2020, respiratory diseases in tested pet cats and dogs were caused by common veterinary pathogens and that SARS‐CoV‐2 infections in dogs and cats are rare.