Swine are a key reservoir host for influenza A viruses (IAVs), with the potential to cause global pandemics in humans. Gaps in surveillance in many of the world's largest swine populations impede our understanding of how novel viruses emerge and expand their spatial range in pigs. Although US swine are intensively sampled, little is known about IAV diversity in Canada's population of ~12 million pigs. By sequencing 168 viruses from multiple regions of Canada, our study reveals that IAV diversity has been underestimated in Canadian pigs for many years. Critically, a new H1 clade has emerged in Canada (H1α-3), with a two-amino acid deletion at H1 positions 146–147, that experienced rapid growth in Manitoba's swine herds during 2014–2015. H1α-3 viruses also exhibit a higher capacity to invade US swine herds, resulting in multiple recent introductions of the virus into the US Heartland following large-scale movements of pigs in this direction. From the Heartland, H1α-3 viruses have disseminated onward to both the east and west coasts of the United States, and may become established in Appalachia. These findings demonstrate how long-distance trading of live pigs facilitates the spread of IAVs, increasing viral genetic diversity and complicating pathogen control. The proliferation of novel H1α-3 viruses also highlights the need for expanded surveillance in a Canadian swine population that has long been overlooked, and may have implications for vaccine design.
Toscana virus (TOSV) is an arthropod-borne phlebovirus within the family Phenuiviridae in the order Bunyavirales. It seems to be an important agent of human meningoencephalitis in the warm season in the Mediterranean area. Because the polymerase of Bunyavirales lacks a capping activity, it cleaves short-capped RNA leaders derived from the host cell, and uses them to initiate viral mRNA synthesis. To determine the size and nucleotide composition of the host-derived RNA leaders, and to elucidate the first steps of TOSV transcription initiation, we performed a high-throughput sequencing of the 5′ end of TOSV mRNAs in infected cells at different times post-infection. Our results indicated that the viral polymerase cleaved the host-capped RNA leaders within a window of 11–16 nucleotides. A single population of cellular mRNAs could be cleaved at different sites to prime the synthesis of several viral mRNA species. The majority of the mRNA resulted from direct priming, but we observed mRNAs resulting from several rounds of prime-and-realign events. Our data suggest that the different rounds of the prime-and-realign mechanism result from the blocking of the template strand in a static position in the active site, leading to the slippage of the nascent strand by two nucleotides when the growing duplex is sorted out from the active site. To minimize this rate-limiting step, TOSV polymerase cleaves preferentially capped RNA leaders after GC, so as to greatly reduce the number of cycles of priming and realignment, and facilitate the separation of the growing duplex.
In vivo imaging is a noninvasive method that enables real-time monitoring of viral infection dynamics in a small animal, which allows a better understanding of viral pathogenesis. In vivo bioluminescence imaging of virus infection is widely used but, despite its advantage over bioluminescence that no substrate administration is required, fluorescence imaging is not used because of severe autofluorescence. Recently, several far-red and near-infrared (NIR) fluorescent proteins (FPs) have been developed and shown to be useful for whole-body fluorescence imaging. Here, we report comparative testing of far-red and NIR FPs in the imaging of rabies virus (RABV) infection. Using the highly neuroinvasive 1088 strain, we generated recombinant RABV that expressed FPs such as Katushka2S, E2-Crimson, iRFP670 or iRFP720. After intracerebral inoculation to nude mice, the 1088 strain expressing iRFP720, the most red-shifted FP, was detected the earliest with the highest signal-to-noise ratio using a filter set for >700 nm, in which the background signal level was very low. Furthermore, we could also track viral dissemination from the spinal cord to the brain in nude mice after intramuscular inoculation of iRFP720-expressing 1088 into the hind limb. Hence, we conclude that the NIR FP iRFP720 used with a filter set for >700 nm is useful for in vivo fluorescence imaging not only for RABV infection but also for other virus infections. Our findings will also be useful for developing dual-optical imaging of virus–host interaction dynamics using bioluminescence reporter mice for inflammation imaging.
Besides the influenza virus (IV), several other viruses are responsible for influenza-like illness (ILI). Although human parechoviruses (HPeVs) and enteroviruses (EVs) may impact on ILI, limited data on their epidemiological characteristics are available. During seven consecutive winter seasons (from 2010–2011 to 2016–2017), within the framework of an influenza surveillance system (InfluNet), 593 respiratory swabs were collected from children ≤5 years of age with ILIs. Molecular detection showed that 58.3 % of swabs were positive for at least one of the viruses under study: 46 % for IV, 13 % for EV and 5.4 % for HPeV. A single virus was identified in 51.3 % of samples while more than one virus was detected in 7 % of the samples. The risk of contracting IV was higher than the risk associated with EV, which in turn was higher than the risk of contracting HPeV. The risk of developing an IV infection was twofold greater in children >3 years than in those ≤3 years, who had higher risk of EV/HPeV infection. The frequency of EV/HPeV-positive swabs increased significantly during the 2016–2017 winter season compared to the previous six seasons. Sixteen EV genotypes were identified belonging to species A and B. HPeV-1 was the most frequently detected genotype, followed by -6 and -3. In this study, IV was mainly responsible for ILI, however EV and HPeV were also involved and particularly affected children ≤3 years of age. Influenza surveillance samples could provide us with valuable insight into the epidemiological features of viruses involved in ILI.