- Volume 103, Issue 6, 2022

Avian rotavirus A (RVA) is one of major enteric pathogens that cause diarrhoea in young avian individuals. Importantly, some of the avian RVA strains of G18P[17] genotype are naturally transmitted to and cause clinical diseases in mammalian species, indicating their potential risks to animal health. Although molecular information on the pathogenesis by avian RVA strains will be useful for estimating their risks, the absence of a reverse genetics (RG) system for these strains has hindered the elucidation of their pathogenic mechanisms. In this study, we aimed to establish an RG system for the avian G18P[17] prototype strain PO-13, which was isolated from a pigeon in Japan in 1983 and was experimentally shown to be pathogenic in suckling mice. Transfection with plasmids expressing 11 genomic RNA segments of the strain resulted in rescue of the infectious virus with an artificially introduced genetic marker on its genome, indicating that an RG system for the PO-13 strain was successfully established. The rescued recombinant strain rPO-13 had biological properties almost identical to those of its wild-type strain (wtPO-13). Notably, both rPO-13 and wtPO-13 induced diarrhoea in suckling mice with similar efficiencies. It was thus demonstrated that the RG system will be useful for elucidating the pathogenic mechanisms of the PO-13 strain at the molecular level. This is the first report of the establishment of an RG system for an avian RVA strain.

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

Constant evolution of influenza A viruses (IAVs) leads to the occurrence of new virus strains, which can cause epidemics and occasional pandemics. Here we compared two medically relevant IAVs, namely A/Hamburg/4/09 (H1N1pdm09) of the 2009 pandemic and the highly pathogenic avian IAV human isolate A/Thailand/1(KAN-1)/2004 (H5N1), for their ability to trigger intracellular phosphorylation patterns using a highly sensitive peptide-based kinase activity profiling approach. Virus-dependent tyrosine phosphorylations of substrate peptides largely overlap between the two viruses and are also strongly overrepresented in comparison to serine/threonine peptide phosphorylations. Both viruses trigger phosphorylations with distinct kinetics by overlapping and different kinases from which many form highly interconnected networks. As approximately half of the kinases forming a signalling hub have no known function for the IAV life cycle, we interrogated selected members of this group for their ability to interfere with IAV replication. These experiments revealed negative regulation of H1N1pdm09 and H5N1 replication by NUAK [novel (nua) kinase] kinases and by redundant ephrin A (EphA) receptor tyrosine kinases.

Costimulatory interactions can be critical in developing immune responses to infectious agents. We recently reported that herpes simplex type 1 (HSV-1) infections of the cornea require a functional CD28-CD80/86 interaction to not only reduce the likelihood of encephalitis, but also to mediate herpetic stromal keratitis (HSK) following viral reactivation. In this same spirit we decided to determine the role that CD137 costimulation plays during HSK. Using both B6-CD137L-/- mice, as well as antagonistic and agonistic antibodies to CD137 we characterize the immune response and to what extent CD137 plays an important role during this disease. Immune responses were measured in both the cornea and in the trigeminal ganglia where the virus forms a latent infection. We demonstrate that CD137 costimulation leads to reduced corneal disease. Interestingly, we observed that lack of CD137 costimulation resulted in significantly reduced CD8+ T expansion and function in the trigeminal ganglia. Finally, we showed that viruses that have been genetically altered to express CD137 display significantly reduced corneal disease, though they did present similar levels of trigeminal infection and peripheral virus production following reactivation of a latent infection. CD137 interactions lead to reduced HSK and are necessary to develop robust trigeminal CD8+ T cell responses.

Koala retrovirus (KoRV) is unique amongst endogenous (inherited) retroviruses in that its incorporation to the host genome is still active, providing an opportunity to study what drives this fundamental process in vertebrate genome evolution. Animals in the southern part of the natural range of koalas were previously thought to be either virus-free or to have only exogenous variants of KoRV with low rates of KoRV-induced disease. In contrast, animals in the northern part of their range universally have both endogenous and exogenous KoRV with very high rates of KoRV-induced disease such as lymphoma. In this study we use a combination of sequencing technologies, Illumina RNA sequencing of ‘southern’ (south Australian) and ‘northern’ (SE QLD) koalas and CRISPR enrichment and nanopore sequencing of DNA of ‘southern’ (South Australian and Victorian animals) to retrieve full-length loci and intregration sites of KoRV variants. We demonstrate that koalas that tested negative to the KoRV pol gene qPCR, used to detect replication-competent KoRV, are not in fact KoRV-free but harbour defective, presumably endogenous, ‘RecKoRV’ variants that are not fixed between animals. This indicates that these populations have historically been exposed to KoRV and raises questions as to whether these variants have arisen by chance or whether they provide a protective effect from the infectious forms of KoRV. This latter explanation would offer the intriguing prospect of being able to monitor and selectively breed for disease resistance to protect the wild koala population from KoRV-induced disease.

CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the ‘shock and kill’ cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5′LTR promoter, making them a promising ‘shock’ strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5′LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3′ half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located −165 to −106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the ‘shock and kill’ cure approach.

During the life cycle of a baculovirus, a crystallized protein matrix, formed by polyhedrin (POLH), is produced. The protein matrix is surrounded by a multilayered protein/carbohydrate envelope, and matrix and envelope together form a mature occlusion body (OB). The polyhedron envelope plays an important role in resistance against adverse external environments. The polyhedron envelope protein (PEP) is the main protein that forms the polyhedron envelope, but the mechanism of formation of the polyhedron envelope is unclear. Here, through immunofluorescence localization observations, we found that PEP interacted with both POLH and P10 during formation of the polyhedron envelope in the late stages of infection, and PEP was also required for P10 incorporation on the surface of OBs. In this process, the phosphorylation of PEP played an important role. PEP was determined to be a phosphorylated protein using the Phos-tag technique, and PK1 was determined to be the phosphokinase of PEP by co-immunoprecipitation and in vitro phosphorylation. Immunofluorescence localization revealed that PEP was continuously phosphorylated by PK1 after PEP entered the nucleus until PEP was correctly packaged on the OB surface. Multi-point mutations of PEP conservative potential phosphorylation sites showed that the simultaneous mutation of S85, T86 and Y92 caused changes in the location of PEP and P10 in the late stages of infection, and resulted in an OB surface that lacked the polyhedron envelope. These data suggested that the phosphorylation of PEP at particular sites, i.e. S85, T86 and Y92, plays an important role in the formation of the polyhedron envelope.