It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2-6 to the next sugar, that avian influenza viruses bind glycans containing the α2-3 linkage, and ...that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2-6(Galβ1-4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2-3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2-3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.
The H1N1 subtype of influenza A virus has caused substantial morbidity and mortality in humans, first documented in the global pandemic of 1918 and continuing to the present day. Despite this disease ...burden, the evolutionary history of the A/H1N1 virus is not well understood, particularly whether there is a virological basis for several notable epidemics of unusual severity in the 1940s and 1950s. Using a data set of 71 representative complete genome sequences sampled between 1918 and 2006, we show that segmental reassortment has played an important role in the genomic evolution of A/H1N1 since 1918. Specifically, we demonstrate that an A/H1N1 isolate from the 1947 epidemic acquired novel PB2 and HA genes through intra-subtype reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the 1951 influenza epidemic may also have been associated with reassortant A/H1N1 viruses. Intra-subtype reassortment therefore appears to be a more important process in the evolution and epidemiology of H1N1 influenza A virus than previously realized.
Identifying SARS-CoV-2 infections through aggressive diagnostic testing remains critical to tracking and curbing the spread of the COVID-19 pandemic. Collection of nasopharyngeal swabs (NPS), the ...preferred sample type for SARS-CoV-2 detection, has become difficult due to the dramatic increase in testing and consequent supply strain. Therefore, alternative specimen types have been investigated that provide similar detection sensitivity with reduced health care exposure and the potential for self-collection. In this study, the detection sensitivity of SARS-CoV-2 in nasal swabs (NS) and saliva was compared to that of NPS using matched specimens from two outpatient cohorts in New York State (total
= 463). The first cohort showed only a 5.4% positivity, but the second cohort (
= 227) had a positivity rate of 41%, with sensitivity in NPS, NS, and saliva of 97.9%, 87.1%, and 87.1%, respectively. Whether the reduced sensitivity of NS or saliva is acceptable must be assessed in the settings where they are used. However, we sought to improve on it by validating a method to mix the two sample types, as the combination of nasal swab and saliva resulted in 94.6% SARS-CoV-2 detection sensitivity. Spiking experiments showed that combining them did not adversely affect the detection sensitivity in either. Virus stability in saliva was also investigated, with and without the addition of commercially available stabilizing solutions. The virus was stable in saliva at both 4°C and room temperature for up to 7 days. The addition of stabilizing solutions did not enhance stability and, in some situations, reduced detectable virus levels.
High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that ...modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.
Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic ...seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.
Since the introduction of the varicella-zoster virus (VZV) vaccine in the United States in 1995, there has been a dramatic decrease in both the number and severity of varicella cases. However, VZV ...surveillance data and information on the VZV clade distribution in central nervous system (CNS) disease and non-CNS disease in New York State is not available. To investigate this, cerebrospinal fluid (CSF) samples from patients with encephalitis or meningitis and non-CSF samples from patients with non-CNS disease manifestations consistent with VZV, collected from 2004 to 2019, were tested with molecular VZV assays. A total of 341 CSF and 1,398 non-CSF samples that tested positive by a VZV-specific real-time PCR assay were further characterized as wild-type or vaccine strain by 3 biallelic real-time PCR assays targeting single nucleotide polymorphism (SNP) markers in open reading frame (ORF) 62. Genotyping was then performed on wild-type strains by conventional PCR and sequencing of 500-bp regions in ORFs 21, 22, and 50. Sequence analysis identified clades 1 to 5 in both sample types with a virtually identical clade distribution between CSF and non-CSF samples. In addition, 19 clade 6 and 13 clade 9 samples were detected in non-CSF samples after implementation of an expanded genotyping scheme, including ORF 29, 38, and 67. These clades were not detected in any CSF samples. Finally, a total of 28 vaccine strains were detected, 25 in the non-CSF samples and 3 in the CSF samples. All three cases of vaccine strain with CNS involvement experienced relatively minor symptoms of aseptic meningitis and fully recovered. These results support the evidence that while the VZV vaccine is capable of causing CNS disease, it is still a rare event and symptoms are typically less severe than those caused by wild-type infection.
Accommodating large increases in sample workloads has presented a major challenge to clinical laboratories during the coronavirus disease 2019 (COVID-19) pandemic. Despite the implementation of ...automated detection systems and previous efficiencies, including barcoding, electronic data transfer, and extensive robotics, capacities have struggled to meet the demand. Sample pooling has been suggested as an additional strategy to address this need. The greatest concern with this approach in clinical settings is the potential for reduced sensitivity, particularly detection failures with weakly positive samples. To investigate this possibility, detection rates in pooled samples were evaluated, with a focus on pools containing weakly positive specimens. Additionally, the frequencies of occurrence of weakly positive samples during the pandemic were reviewed. Weakly positive specimens, with threshold cycle (
) values of 33 or higher, were detected in 95% of 60 five-sample pools but only 87% of 39 nine-sample pools. The proportion of positive samples with very low viral loads rose markedly during the first few months of the pandemic, peaking in June, decreasing thereafter, and remaining level since August. At all times, weakly positive specimens comprised a significant component of the sample population, ranging from 29% to >80% for
values above 31. In assessing the benefits of pooling strategies, however, other aspects of the testing process must be considered. Accessioning, result data management, electronic data transfer, reporting, and billing are not streamlined and may be complicated by pooling procedures. Therefore, the impact on the entire laboratory process needs to be carefully assessed prior to implementing such a strategy.
The error rate of the RNA-dependent RNA polymerase (RdRp) of RNA viruses is important in maintaining genetic diversity for viral adaptation and fitness. Numerous studies have shown that ...mutagen-resistant RNA virus variants display amino acid mutations in the RdRp and other replicase subunits, which in turn exhibit an altered fidelity phenotype affecting viral fitness, adaptability and pathogenicity. St. Louis encephalitis virus (SLEV), like its close relative West Nile virus, is a mosquito-borne flavivirus that has the ability to cause neuroinvasive disease in humans. Here, we describe the successful generation of multiple ribavirin-resistant populations containing a shared amino acid mutation in the SLEV RdRp (E416K). These E416K mutants also displayed resistance to the antiviral T-1106, an RNA mutagen similar to ribavirin. Structural modelling of the E416K polymerase mutation indicated its location in the pinky finger domain of the RdRp, distant from the active site. Deep sequencing of the E416K mutant revealed lower genetic diversity than wild-type SLEV after growth in both vertebrate and invertebrate cells. Phenotypic characterization showed that E416K mutants displayed similar or increased replication in mammalian cells, as well as modest attenuation in mosquito cells, consistent with previous work with West Nile virus high-fidelity variants. In addition, attenuation was limited to mosquito cells with a functional RNA interference response, suggesting an impaired capacity to escape RNA interference could contribute to attenuation of high-fidelity variants. Our results provide increased evidence that RNA mutagen resistance arises through modulation of the RdRp and give further insight into the consequences of altered fidelity of flaviviruses.
The emergence of recombinant viruses is a threat to public health, as recombination may integrate variant-specific features that together result in escape from treatment or immunity. The selective ...advantages of recombinant SARS-CoV-2 isolates over their parental lineages remain unknown. We identified a Delta-Omicron (AY.45-BA.1) recombinant in an immunosuppressed transplant recipient treated with monoclonal antibody Sotrovimab. The single recombination breakpoint is located in the spike N-terminal domain adjacent to the Sotrovimab binding site. While Delta and BA.1 are sensitive to Sotrovimab neutralization, the Delta-Omicron recombinant is highly resistant. To our knowledge, this is the first described instance of recombination between circulating SARS-CoV-2 variants as a functional mechanism of resistance to treatment and immune escape.
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•Delta-Omicron recombinant in an immunosuppressed individual treated with Sotrovimab•Delta and Omicron (BA.1) are sensitive to Sotrovimab, but recombinant is resistant•Spike E340D mutation confers resistance to Sotrovimab and vaccine sera•Delta-Omicron recombinant is resistant to Sotrovimab irrespective of E340D
Biological sciences; Immunology; Immunity.
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