For diverse viruses, cellular infection with single vs. multiple virions can yield distinct biological outcomes. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells. Here, we sought to uncover the viral processes underlying this phenotype. We found that the need for multiple infection maps to amino acid 26K of the viral PA protein. PA 26K suppresses endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. In the context of the higher functioning PA 26E, inhibition of PA using baloxavir acid augments reliance on multiple infection. Together, these data suggest a model in which sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral ribonucleoprotein templates to the cell. More broadly, the finding that deficiency in a core viral function is ameliorated through multiple infection suggests that the fitness effects of many viral mutations are likely to be modulated by multiplicity of infection, such that the shape of fitness landscapes varies with viral densities.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.

Transmission efficiency is a critical factor determining the size of an outbreak of infectious disease. Indeed, the propensity of SARS-CoV-2 to transmit among humans precipitated and continues to sustain the COVID-19 pandemic. Nevertheless, the number of new cases among contacts is highly variable and underlying reasons for wide-ranging transmission outcomes remain unclear. Here, we evaluated viral spread in golden Syrian hamsters to define the impact of temporal and environmental conditions on the efficiency of SARS-CoV-2 transmission through the air. Our data show that exposure periods as brief as one hour are sufficient to support robust transmission. However, the timing after infection is critical for transmission success, with the highest frequency of transmission to contacts occurring at times of peak viral load in the donor animals. Relative humidity and temperature had no detectable impact on transmission when exposures were carried out with optimal timing and high inoculation dose. However, contrary to expectation, trends observed with sub-optimal exposure timing and lower inoculation dose suggest improved transmission at high relative humidity or high temperature. In sum, among the conditions tested, our data reveal the timing of exposure to be the strongest determinant of SARS-CoV-2 transmission success and implicate viral load as an important driver of transmission.

16The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its 17 emergence into human populations. To elucidate the genetic contributions of the M segment to 18 host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that 19 carry avian-or human-derived M segments. Avian, but not human, M segments restricted viral 20 growth and transmission in mammalian model systems, and the restricted growth correlated 21 with increased expression of M2 relative to M1. M2 overexpression was associated with 22 intracellular accumulation of autophagosomes, which was alleviated by interference of the viral 23 proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M 24 mRNA through alternative splicing, we separated synonymous and non-synonymous changes 25 that differentiate human and avian M segments and found that dysregulation of gene expression 26 leading to M2 overexpression diminished replication, irrespective of amino acid composition of 27 M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that 28 expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) 29 determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment 30 gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated 31 dysregulation of vesicular homeostasis. 33Author summary 34 Influenza A virus (IAV) pandemics arise when a virus adapted to a non-human host overcomes 35 species barriers to successfully infect humans and sustain human-to-human transmission. To 36 gauge the adaptive potential and therefore pandemic risk posed by a particular IAV, it is critical 37 to understand the mechanisms underlying viral adaptation to human hosts. Here, we focused on 38 the role of one of IAV's eight gene segments, the M segment, in host adaptation. Comparing the 39 growth of IAVs with avian-and human-derived M segments in avian and mammalian systems 40 revealed that the avian M segment restricts viral growth specifically in mammalian cells. We 41 3 show that the mechanism underlying this host range phenotype is a dysregulation of viral gene 42 expression when the avian IAV M segment is transcribed in mammalian cells. In particular, 43 excess production of the M2 protein results in viral interference with cellular functions on which 44 the virus relies. Our results therefore reveal that the use of cellular machinery to control viral 45 gene expression leaves the virus vulnerable to over-or under-production of critical viral gene 46 products in a new host species. 47Changes to viral receptor specificity and polymerase function necessary to overcome host 67 species barriers have been well documented [7][8][9][10][11], but it is clear that additional features of 68 avian IAVs restrict their growth in mammalian systems [12][13][14]. Seminal studies, performed in 69 the 1970s, indicated a potential role for the M segment. Avian IAVs were shown to express low 70...