ORCID Profile
0000-0001-5442-3883
Current Organisation
Cornell University
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Publisher: Cold Spring Harbor Laboratory
Date: 27-06-2019
DOI: 10.1101/683516
Abstract: Influenza A viruses have regularly jumped to new hosts to cause epidemics or pandemics, an evolutionary process that involves variation in the viral traits necessary to overcome host barriers and facilitate transmission. Mice are not a natural host for influenza virus, but are frequently used as models in studies of pathogenesis, often after multiple passages to achieve higher viral titers that result in clinical disease such as weight loss or death. Here we examine the processes of influenza A virus infection and evolution in mice by comparing deep sequence variation of a human H1N1 pandemic virus, a seasonal H3N2 virus, and a H3N2 canine influenza virus during experimental passage. We also compared replication and sequence variation in wild-type mice expressing N-glycolylneuraminic acid (Neu5Gc) with that seen in mice expressing only N-acetylneuraminic acid (Neu5Ac). Viruses derived from plasmids were propagated in MDCK cells and then passaged in mice up to four times. Full genome deep sequencing of the plasmids, cultured viruses, and viruses from mice at various passages revealed only small numbers of mutational changes. The H3N2 canine influenza virus showed increases in frequency of sporadic mutations in the PB2, PA, and NA segments. The H1N1 pandemic virus grew well in mice, and while it exhibited the maintenance of some minority mutations, there was no clear adaptive evolution. The H3N2 seasonal virus did not establish in the mice. Finally, there were no clear sequence differences associated with the presence or absence of Neu5Gc. Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals, but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not effect influenza evolution in this model. Overall this shows that mice provide a useful animal model for influenza, but that host passage evolution will vary depending on the virus being tested.
Publisher: Cold Spring Harbor Laboratory
Date: 23-02-2020
DOI: 10.1101/2020.02.20.959015
Abstract: New methods for deep sequence analysis provide an opportunity to follow the emergence and dynamics of virus mutations in real time. Although viruses are commonly grown in cell culture for research and for vaccine development, the cells used to grow the virus are often not derived from the same tissue or even the same host that the virus naturally replicates in. The selective pressures of culturing virus in vitro are still only partially understood. MDCK cells are the standard cell for growing influenza viruses yet are derived from the epithelium of the canine kidney and are also heterogenous. We passaged human H3N2, H1N1 pandemic, and canine H3N2 influenza A viruses (IAV) in different lineages of MDCK cells, as well as lines engineered to express variant Sia receptors, including α2,3- and α2,6-linkages or N -glycolylneuraminic acid (Neu5Gc) or N -acetylneuraminic acid (Neu5Ac) forms. MDCK-Type II cells had lower infection efficiency and virus production, and infection appeared more dependent on protease activation of the virus. When viruses were passaged in the different cells, they exhibited only small numbers of consensus-level mutations, and most were within the HA gene. Both human IAVs showed selection for single nucleotide minority variants in the HA stem across cell types, as well as low frequency variants in the HA receptor binding site of virus passaged in cells expressing Neu5Gc. Canine H3N2 also showed minority variants near the receptor-binding site in cells expressing Neu5Gc and also in those expressing α 2,6-linkages. The genetic variation and adaptability of viruses are fundamental properties that allow their evolutionary success in the face of differing host environments and immune responses. The growth of viruses in cell culture is widely used for their study and for preparing vaccines. However, the selection pressures that cell passaging imposes on viruses are often poorly understood. We used deep sequence analysis to define, in detail, how three different influenza A viruses respond to passaging in different lineages of canine MDCK cells that are commonly used for their growth, as well as in variant cells engineered to express different forms of their cell surface receptor, sialic acid. This analysis revealed that most mutations occur in the HA gene and few sequence changes in the virus population reached high proportions. This is relevant for understanding the selective pressures of virus growth in cell culture and how it shapes evolutionary patterns.
Publisher: Wiley
Date: 27-06-2021
DOI: 10.1111/JEB.13890
Abstract: Viruses are often cultured in cell lines for research and vaccine development, and those often differ from the natural hosts or tissues. Cell lines can also differ in the presence of virus receptors, such as the sialic acid (Sia) receptors used by influenza A viruses (IAV), which can vary in linkage (α2,3‐ or α2,6‐linkage) and form ( N ‐glycolylneuraminic acid [Neu5Gc] or N ‐acetylneuraminic acid [Neu5Ac]). The selective pressures resulting from passaging viruses in cell types with host‐specific variations in viral receptors are still only partially understood. IAV are commonly cultured in MDCK cells which are both derived from canine kidney tubule epithelium and inherently heterogeneous. MDCK cells naturally present Neu5Ac and α2,3‐linked Sia forms. Here, we examine natural MDCK variant lineages, as well as engineered variants that synthesize Neu5Gc and/or α2,6‐linkages. We determined how viral genetic variation occurred within human H3N2, H1N1 pandemic and canine H3N2 IAV populations when serially passaged in MDCK cell lines that vary in cell type (MDCK‐Type I or MDCK‐Type II clones) and in Sia display. Deep sequencing of viral genomes showed small numbers of consensus‐level mutations, mostly within the hemagglutinin (HA) gene. Both human IAV showed variants in the HA stem and the HA receptor‐binding site of populations passaged in cells displaying Neu5Gc. Canine H3N2 showed variants near the receptor‐binding site when passaged in cells displaying Neu5Gc or α2,6‐linkages. Viruses replicated to low titres in MDCK‐Type II cells, suggesting that not all cell types in heterogeneous MDCK cell populations are equally permissive to infection.
Publisher: Cold Spring Harbor Laboratory
Date: 23-03-2023
DOI: 10.1101/2023.03.22.533763
Abstract: Cross-species virus transmission events can lead to dire public health emergencies in the form of epidemics and pandemics. One ex le in animals is the emergence of the H3N8 equine influenza virus (EIV), first isolated in 1963 in Miami, Florida, USA, after emerging among horses in South America. In the early 21 st century the American lineage of EIV erged into two ‘Florida’ clades that persist today, while an EIV transferred to dogs around 1999 and gave rise to the H3N8 canine influenza virus (CIV), first reported in 2004. Here, we compare CIV in dogs and EIV in horses to reveal their host-specific evolution, to determine the sources and connections between significant outbreaks, and to gain insight into the factors controlling their different evolutionary fates. H3N8 CIV only circulated in North America, was geographically restricted after the first few years, and went extinct in 2016. Of the two EIV Florida clades, clade 1 circulates widely and shows frequent transfers between the USA and South America, Europe and elsewhere, while clade 2 was globally distributed early after it emerged, but since about 2018 has only been detected in Central Asia. Any potential zoonotic threat of these viruses to humans can only be determined with an understanding of its natural history and evolution. Our comparative analysis of these three viral lineages reveals distinct patterns and rates of sequence variation yet with similar overall evolution between clades, suggesting epidemiological intervention strategies for possible eradication of H3N8 EIV. (242 words) The emergence of viruses in new hosts is a threat to human and animal health. The H3N8 equine influenza virus (EIV) emerged in 1963 by transfer of an avian influenza virus, and the H3N8 canine influenza virus (CIV) subsequently emerged in 1999 when EIV transferred to dogs. H3N8 CIV persistently circulated in only a few locations in the USA, and has not been detected since 2016. In the same period H3N8 EIV has circulated as two separate clades, one in North America and other regions of the world, while the other currently appears to be found only in Central Asia. By comparing the hosts, epidemiology, and evolution of these influenza viruses we explain how these lineages had different evolutionary fates, and show why elucidating these evolutionary processes is key to understanding zoonotic disease and viral emergence. (137 words)
Publisher: Cold Spring Harbor Laboratory
Date: 19-01-2023
DOI: 10.1101/2023.01.18.524668
Abstract: Canine parvovirus (CPV) is a small non-enveloped single-stranded DNA virus that causes serious diseases in dogs worldwide. The original strain of the virus (CPV-2) emerged in dogs during the late-1970s due to a host range switch of a virus similar to the feline panleukopenia virus (FPV) that infected another host. The virus that emerged in dogs had altered capsid receptor- and antibody-binding sites, with some changes affecting both functions. Further receptor and antibody binding changes arose when the virus became better adapted to dogs or to other hosts. Here, we use in vitro selection and deep sequencing to reveal how two antibodies with known interactions select for escape mutations in CPV. The antibodies bind two distinct epitopes, and one largely overlaps the host receptor binding site. We also engineered antibody variants with altered binding structures. Viruses were passaged with the wild type or mutated antibodies, and their genomes deep sequenced during the selective process. A small number of mutations were detected only within the capsid protein gene during the first few passages of selection, and most sites remained polymorphic or were slow to go to fixation. Mutations arose both within and outside the antibody binding footprints on the capsids, and all avoided the TfR-binding footprint. Many selected mutations matched those that have arisen in the natural evolution of the virus. The patterns observed reveal the mechanisms by which these variants have been selected in nature and provide a better understanding of the interactions between antibody and receptor selections. Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system. Here, we use an in vitro model system and deep genome sequencing to reveal the mutations that arise in the virus genome during selection by each of two monoclonal antibodies or their engineered variants. High-resolution structures of each of the Fab: capsid complexes revealed their binding interactions. The engineered forms of the wild-type antibodies or mutant forms allowed us to examine how changes in antibody structure influence the mutational selection patterns seen in the virus. The results shed light on the processes of antibody binding, neutralization escape, and receptor binding, and likely have parallels for many other viruses.
Publisher: American Society for Microbiology
Date: 29-06-2023
DOI: 10.1128/JVI.00090-23
Abstract: Antibodies protect animals against infection by many different viruses and other pathogens, and we are gaining new information about the epitopes that induce antibody responses against viruses and the structures of the bound antibodies. However, less is known about the processes of antibody selection and antigenic escape and the constraints that apply in this system.
Publisher: American Society for Microbiology
Date: 07-2015
DOI: 10.1128/JVI.00521-15
Abstract: The A/H3N8 canine influenza virus (CIV) emerged from A/H3N8 equine influenza virus (EIV) around the year 2000 through the transfer of a single virus from horses to dogs. We defined and compared the biological properties of EIV and CIV by examining their genetic variation, infection, and growth in different cell cultures, receptor specificity, hemagglutinin (HA) cleavage, and infection and growth in horse and dog tracheal explant cultures. Comparison of sequences of viruses from horses and dogs revealed mutations that may be linked to host adaptation and tropism. We prepared infectious clones of representative EIV and CIV strains that were similar to the consensus sequences of viruses from each host. The rescued viruses, including HA and neuraminidase (NA) double reassortants, exhibited similar degrees of long-term growth in MDCK cells. Different host cells showed various levels of susceptibility to infection, but no differences in infectivity were seen when comparing viruses. All viruses preferred α2-3- over α2-6-linked sialic acids for infections, and glycan microarray analysis showed that EIV and CIV HA-Fc fusion proteins bound only to α2-3-linked sialic acids. Cleavage assays showed that EIV and CIV HA proteins required trypsin for efficient cleavage, and no differences in cleavage efficiency were seen. Inoculation of the viruses into tracheal explants revealed similar levels of infection and replication by each virus in dog trachea, although EIV was more infectious in horse trachea than CIV. IMPORTANCE Influenza A viruses can cross species barriers and cause severe disease in their new hosts. Infections with highly pathogenic avian H5N1 virus and, more recently, avian H7N9 virus have resulted in high rates of lethality in humans. Unfortunately, our current understanding of how influenza viruses jump species barriers is limited. Our aim was to provide an overview and biological characterization of H3N8 equine and canine influenza viruses using various experimental approaches, since the canine virus emerged from horses approximately 15 years ago. We showed that although there were numerous genetic differences between the equine and canine viruses, this variation did not result in dramatic biological differences between the viruses from the two hosts, and the viruses appeared phenotypically equivalent in most assays we conducted. These findings suggest that the cross-species transmission and adaptation of influenza viruses may be mediated by subtle changes in virus biology.
Publisher: American Society for Microbiology
Date: 12-2019
DOI: 10.1128/JVI.01039-19
Abstract: Mice are commonly used as a model to study the growth and virulence of influenza A viruses in mammals but are not a natural host and have distinct sialic acid receptor profiles compared to humans. Using experimental infections with different subtypes of influenza A virus derived from different hosts, we found that evolution of influenza A virus in mice did not necessarily proceed through the linear accumulation of host-adaptive mutations, that there was variation in the patterns of mutations detected in each repetition, and that the mutation dynamics depended on the virus examined. In addition, variation in the viral receptor, sialic acid, did not affect influenza virus evolution in this model. Overall, our results show that while mice provide a useful animal model for influenza virus pathology, host passage evolution will vary depending on the specific virus tested.
Publisher: Oxford University Press (OUP)
Date: 07-2023
DOI: 10.1093/VE/VEAD052
No related grants have been discovered for Brian Wasik.