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Background Eastern equine encephalitis virus is a mosquito-borne alphavirus responsible for unpredictable outbreaks of severe neurologic disease in animals and humans. While most human infections are asymptomatic or clinically nonspecific, a minority of patients develops encephalitic disease, a devastating illness with a mortality rate of ≥30%. No treatments are known to be effective. Eastern equine encephalitis virus infection is rare in the United States, with an annual average nationwide incidence of 7 cases between 2009 and 2018. However, in 2019, 38 cases were confirmed nationwide, including 10 in Michigan. Methods Data from 8 cases identified by a regional network of physicians in southwest Michigan were abstracted from clinical records. Clinical imaging and histopathology were aggregated and reviewed. Results Patients were predominantly older adults (median age, 64 years), and all were male. Results of initial arboviral cerebrospinal fluid serology were frequently negative, and diagnosis was not made until a median of 24.5 days (range, 13–38 days) after presentation, despite prompt lumbar punctures in all patients. Imaging findings were dynamic and heterogeneous, with abnormalities of the thalamus and/or basal ganglia, and prominent pons and midbrain abnormalities were displayed in 1 patient. Six patients died, 1 survived the acute illness with severe neurologic sequelae, and 1 recovered with mild sequelae. A limited postmortem examination revealed diffuse meningoencephalitis, neuronophagia, and focal vascular necrosis. Conclusions Eastern equine encephalitis is a frequently fatal condition whose diagnosis is often delayed, and for which no effective treatments are known. Improved diagnostics are needed to facilitate patient care and encourage the development of treatments.

Background Guidelines recommend use of tocilizumab (TCZ), an inhibitor of pro-inflammatory IL-6 signaling, for hospitalized patients with progressive severe or critical Coronavirus disease 2019 (COVID-19). The incidence of infectious complications following the use of TCZ for COVID-19 has not been well-described. Methods We conducted a retrospective, observational matched cohort study of severely ill, hospitalized adult patients with confirmed COVID-19 who were treated with TCZ between 2/24/2021 and 6/1/2021. The intervention group, comprised of patients who received a single dose of TCZ, was matched based on c-reactive protein (CRP) and fraction of inspired oxygen (FiO2) with a control group who did not receive TCZ, and were hospitalized between 10/12/2020 and 3/6/2021. The primary outcome of the study was diagnosis of a bacterial or fungal infection after day 3 of the index hospitalization. Secondary outcomes included all-cause mortality during the study period and length of stay. Results 75 patients who received TCZ were identified during the study period, and matched 1:1 with 75 control patients. Baseline CRP and FiO2 were similar between groups, while the median age in the TCZ group was younger (61 versus 71 years), reflecting the epidemiology of the outbreak during the TCZ and control study periods. 15 bacterial and fungal infections were identified in the TCZ group (20.0%) and 4 (5.3%) infections in the control group (p = 0.012). The majority of infections in both groups were bacterial, with a disproportionate number of bloodstream infections in the TCZ group [7 (9.3%) vs 2(2.6%); p = 0.166]. 28 patients (37.3%) died in the TCZ group compared to 39 (52.0%) in the matched control (p = 0.068). Median time to discharge was similar between TCZ and control patients (11 versus 12 days; 95% CI -2,2). Conclusion Secondary infections in adult patients with severe and critical COVID-19 were more common in patients who had received TCZ. Prospective studies are needed to confirm and further describe this association. Disclosures All Authors: No reported disclosures

Background Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract has raised concerned for potential damaging effects on function. Animal studies have demonstrated that ZIKV virus can infect and damage the testis and epididymis, and these results has been correlated to lower sperm counts in ZIKV-infected humans. The prostate plays a vital role in the male reproductive tract, with acute and chronic prostatitis linked to male infertility. Methods In this study, we evaluated the effects of ZIKV virus on the prostate in mice and nonhuman primates. Results In mice, ZIKV infected the prostate and triggered inflammation that persisted even after virus clearance. Evidence of chronic prostatitis associated with ZIKV infection remained for several months. Similar histological findings were observed in the prostate of ZIKV-infected rhesus macaques. Conclusions These studies establish that ZIKV replicates in the prostate and can cause acute and chronic inflammatory and proliferative changes in mouse and nonhuman primate models.

Infections with dengue virus (DENV) and Zika virus (ZIKV) can induce cross-reactive antibody responses. Two immunodominant epitopes-one to precursor membrane protein and one to the fusion loop epitope on envelope (E) protein-are recognized by cross-reactive antibodies1-3 that are not only poorly neutralizing, but can also promote increased viral replication and disease severity via Fcγ receptor-mediated infection of myeloid cells-a process termed antibody-dependent enhancement (ADE)1,4,5. ADE is a significant concern for both ZIKV and DENV vaccines as the induction of poorly neutralizing cross-reactive antibodies may prime an individual for ADE on natural infection. In this report, we describe the design and production of covalently stabilized ZIKV E dimers, which lack precursor membrane protein and do not expose the immunodominant fusion loop epitope. Immunization of mice with ZIKV E dimers induces dimer-specific antibodies, which protect against ZIKV challenge during pregnancy. Importantly, the ZIKV E-dimer-induced response does not cross-react with DENV or induce ADE of DENV infection.

Zika virus (ZIKV) caused an epidemic of congenital malformations in 2015–2016. Although many vaccine candidates have been generated, few have demonstrated efficacy against congenital ZIKV infection. Here, we evaluated lipid-encapsulated messenger RNA (mRNA) vaccines and a DNA plasmid vaccine encoding the prM-E genes of ZIKV in mouse models of congenital infection. Although the DNA vaccine provided comparable efficacy against vertical transmission of ZIKV, the mRNA vaccines, including one that minimizes antibody-dependent enhancement of infection, elicited higher levels of antigen-specific long-lived plasma cells and memory B cells. Despite the induction of robust neutralizing antibody titers by all vaccines, breakthrough seeding of the placenta and fetal head was observed in a small subset of type I interferon signaling–deficient immunocompromised dams. In comparison, evaluation of one of the mRNA vaccines in a human STAT2-knockin transgenic immunocompetent mouse showed complete protection against congenital ZIKV transmission. These data will inform ongoing human ZIKV vaccine development efforts and enhance our understanding of the correlates of vaccine-induced protection.

Tick-borne infections represent a significant health risk each year in the United States. Immunocompromised patients are typically at risk of more severe disease manifestations than their immunocompetent counterparts. Here we report a case of a newly emerging phlebovirus, Heartland virus, in a heart transplant recipient.

A G7P5A1 woman in her 40s presented to the emergency department at 37 weeks 3 days’ estimated gestational age (EGA) with headache, lip tingling and several days of difficulty speaking. Physical examination demonstrated bilateral facial weakness in a peripheral distribution, as well as decreased corneal reflexes and cervical lymphadenopathy. Routine fourth generation HIV screening had previously been negative at 14 and 28 weeks’ EGA. Brain MRI was unremarkable, and lumbar puncture disclosed a low-grade, mononuclear cerebrospinal fluid pleocytosis; the patient was treated supportively. She returned for induction of labour at 39 weeks, at which time HIV infection was unexpectedly diagnosed. While unilateral idiopathic peripheral facial paralysis is associated with the third trimester of pregnancy and the early postpartum period, bilateral facial paralysis is rare and should prompt work-up for an underlying systemic cause, such as HIV infection.

Background Eastern Equine Encephalitis Virus (EEEV) is a mosquito-borne alphavirus responsible for unpredictable outbreaks of severe neurologic disease in humans. While the vast majority of human EEEV infections are either asymptomatic or clinically nonspecific, a minority of patients develops neuroinvasive disease (EEE), which is a devastating illness with a mortality of at least 30%. No treatments are known to be effective. EEEV infection is relatively rare in the United States, with an annual average nationwide incidence of 7 cases between 2009 and 2018. However, 2019 was an exceptionally active year for human EEEV disease, yielding 38 nationwide confirmed cases, including 10 in Michigan, comprising the state’s largest outbreak to date. Methods EEE cases were identified by a regional network of physicians. Cases were defined by presentation with clinical symptoms of encephalitis, and by identification of EEEV IgM antibodies or RNA in cerebrospinal fluid (CSF), or EEEV-specific IgM in serum as confirmed by plaque reduction neutralization test. Radiographic images were evaluated and clinical data abstracted through chart review and clinical follow-up where possible. Results Records from 7 patients were identified and reviewed. The median age was 64, with a male predominance, and all presented in August. Notably, commercial arboviral CSF serology was uniformly negative on the initial CSF sample, and diagnosis was not made until a mean of 23 days (range: 12–38 days) after presentation. Testing in public health laboratories yielded the diagnosis in 5 out of 7 cases. Imaging findings were heterogeneous, but most patients exhibited abnormal findings in the thalamus and/or basal ganglia, and one patient displayed prominent pons and midbrain abnormalities. 4 patients died, while 2 patients survived with severe neurologic sequelae, and 1 patient recovered without sequelae. One patient underwent a limited postmortem examination, which revealed diffuse meningoencephalitis and focal vascular necrosis. Conclusion EEE is a frequently fatal condition whose diagnosis is often delayed, and for which no effective treatments are known. Improved diagnostics are needed to facilitate further clinical studies of EEE and encourage the development of potential therapies. Disclosures All Authors: No reported disclosures

The PA-X protein of influenza A virus has roles in host cell shut-off and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression compared with PA-Xs from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X-ORF, had little effect on the infectious virus titre of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens’ eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology; effects that were more pronounced in the PR8 strain than the T/E reassortant, despite the low shut-off activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of HA to NP and M1 compared to their WT counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and NA segments from H1N1 2009 pandemic viruses or an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbour a function unrelated to host cell shut-off and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCEInfluenza A virus is a widespread pathogen that affects both man and a variety of animal species, causing regular epidemics and sporadic pandemics with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans, mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs., The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens’ eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses. IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with nucleic acid or inactivated viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.

Summary Although Zika virus (ZIKV)-induced congenital disease occurs more frequently during early stages of pregnancy, its basis remains undefined. Using established type I interferon (IFN)-deficient mouse models of ZIKV transmission in utero, we found that the placenta and fetus were more susceptible to ZIKV infection at earlier gestational stages. Whereas ZIKV infection at embryonic day 6 (E6) resulted in placental insufficiency and fetal demise, infections at midstage (E9) resulted in reduced cranial dimensions, and infection later in pregnancy (E12) caused no apparent fetal disease. In addition, we found that fetuses lacking type III IFN-λ signaling had increased ZIKV replication in the placenta and fetus when infected at E12, and reciprocally, treatment of pregnant mice with IFN-λ2 reduced ZIKV infection. IFN-λ treatment analogously diminished ZIKV infection in human midgestation fetal- and maternal-derived tissue explants. Our data establish a model of gestational stage dependence of ZIKV pathogenesis and IFN-λ-mediated immunity at the maternal-fetal interface.

Background Zika virus (ZIKV) caused an epidemic of microcephaly and congenital malformations in 2015–2016, prompting the development of ZIKV vaccines. Plasmid DNA and modified mRNA lipid nanoparticle-encapsulated (mRNA-LNP) vaccines were among the first to reach human clinical trials, where their evaluation is ongoing. Few studies have evaluated vaccine efficacy in the setting of infection during pregnancy, and there is an open question around antibody-dependent enhancement (ADE) of flaviviral disease due to cross-reactive fusion loop epitope (FLE) antibodies. Methods Female C57BL/6J mice and human STAT2 knock-in (hSTAT2-KI) mice were immunized with plasmid DNA (VRC5283) or mRNA-LNP (Moderna Inc.) vaccines encoding the ZIKV prM-E genes. Antibody responses were assayed, and immunized mice were mated and WT mice were transiently immunocompromised by administration of interferon blocking antibody, followed by ZIKV challenge. 1 week post-infection, ZIKV burden was measured via qRT-PCR. ZIKV-specific memory B cell (MBC), long-lived plasma cell (LLPC), and CD8+ T cell vaccine responses were also assayed. Results VRC5283 and mRNA-LNP vaccines were highly immunogenic, eliciting serum neutralizing EC50 responses >1:10,000, and markedly reduced placental ZIKV burden and fetal transmission. An improved mRNA-LNP construct with higher immunogenicity correlated with reduced placental viral burden. Significantly, an FLE-mutant mRNA-LNP vaccine yielded comparable EC50 responses without compromising vaccine efficacy; sera from these mice did not enhance dengue virus infection in vitro. Both VRC5283 and mRNA-LNP vaccines elicited MBC, LLPC, and CD8+ T cell responses, although MBC and LLPC responses were greater after mRNA-LNP immunization. Surprisingly, low-level ZIKV infection of the placenta and a minority of fetal heads were observed despite robust neutralizing antibody responses, which was not seen in the immunocompetent hSTAT2-KI model. Conclusion Nucleic acid vaccines were highly immunogenic and protective against vertical ZIKV transmission during pregnancy in mice. These data support and inform the ongoing clinical development of these vaccines in humans. Disclosures All Authors: No reported Disclosures.

The 1918 influenza pandemic caused over 40 million deaths worldwide, with 675,000 deaths in the United States alone. Studies in several experimental animal models showed that 1918 influenza virus infection resulted in severe lung pathology associated with dysregulated immune and cell death responses. To determine if reactive oxygen species produced by host inflammatory responses play a central role in promoting severity of lung pathology, we treated 1918 influenza virus-infected mice with the catalytic catalase/superoxide dismutase mimetic, salen-manganese complex EUK-207 beginning 3 days postinfection. Postexposure treatment of mice infected with a lethal dose of the 1918 influenza virus with EUK-207 resulted in significantly increased survival and reduced lung pathology without a reduction in viral titers. In vitro studies also showed that EUK-207 treatment did not affect 1918 influenza viral replication. Immunohistochemical analysis showed a reduction in the detection of the apoptosis marker cleaved caspase-3 and the oxidative stress marker 8-oxo-2'-deoxyguanosine in lungs of EUK-207-treated animals compared to vehicle controls. High-throughput sequencing and RNA expression microarray analysis revealed that treatment resulted in decreased expression of inflammatory response genes and increased lung metabolic and repair responses. These results directly demonstrate that 1918 influenza virus infection leads to an immunopathogenic immune response with excessive inflammatory and cell death responses that can be limited by treatment with the catalytic antioxidant EUK-207.

Zika virus infection during pregnancy can cause congenital abnormities or fetal demise. The persistence of Zika virus in the male reproductive system poses a risk of sexual transmission. Here we demonstrate that live-attenuated Zika virus vaccine candidates containing deletions in the 3′ untranslated region of the Zika virus genome (ZIKV-3′UTR-LAV) prevent viral transmission during pregnancy and testis damage in mice, as well as infection of nonhuman primates. After a single-dose vaccination, pregnant mice challenged with Zika virus at embryonic day 6 and evaluated at embryonic day 13 show markedly diminished levels of viral RNA in maternal, placental, and fetal tissues. Vaccinated male mice challenged with Zika virus were protected against testis infection, injury, and oligospermia. A single immunization of rhesus macaques elicited a rapid and robust antibody response, conferring complete protection upon challenge. Furthermore, the ZIKV-3′UTR-LAV vaccine candidates have a desirable safety profile. These results suggest that further development of ZIKV-3′UTR-LAV is warranted for humans., Zika virus infection can result in congenital disorders and cause disease in adults, and there is currently no approved vaccine. Here Shan et al. show that a single dose of a live-attenuated Zika vaccine prevents infection, testis damage and transmission to the fetus during pregnancy in different animal models.

Two alleles of segment 8 (NS) circulate in nonchiropteran influenza A viruses. The A allele is found in avian and mammalian viruses, but the B allele is viewed as being almost exclusively found in avian viruses. This might reflect the fact that one or both of its encoded proteins (NS1 and NEP) are maladapted for replication in mammalian hosts. To test this, a number of clade A and B avian virus-derived NS segments were introduced into human H1N1 and H3N2 viruses. In no case was the peak virus titer substantially reduced following infection of various mammalian cell types. Exemplar reassortant viruses also replicated to similar titers in mice, although mice infected with viruses with the avian virus-derived segment 8s had reduced weight loss compared to that achieved in mice infected with the A/Puerto Rico/8/1934 (H1N1) parent. In vitro, the viruses coped similarly with type I interferons. Temporal proteomics analysis of cellular responses to infection showed that the avian virus-derived NS segments provoked lower levels of expression of interferon-stimulated genes in cells than wild type-derived NS segments. Thus, neither the A nor the B allele of avian virus-derived NS segments necessarily attenuates virus replication in a mammalian host, although the alleles can attenuate disease. Phylogenetic analyses identified 32 independent incursions of an avian virus-derived A allele into mammals, whereas 6 introductions of a B allele were identified. However, A-allele isolates from birds outnumbered B-allele isolates, and the relative rates of Aves-to-Mammalia transmission were not significantly different. We conclude that while the introduction of an avian virus segment 8 into mammals is a relatively rare event, the dogma of the B allele being especially restricted is misleading, with implications in the assessment of the pandemic potential of avian influenza viruses. IMPORTANCE Influenza A virus (IAV) can adapt to poultry and mammalian species, inflicting a great socioeconomic burden on farming and health care sectors. Host adaptation likely involves multiple viral factors. Here, we investigated the role of IAV segment 8. Segment 8 has evolved into two distinct clades: the A and B alleles. The B-allele genes have previously been suggested to be restricted to avian virus species. We introduced a selection of avian virus A- and B-allele segment 8s into human H1N1 and H3N2 virus backgrounds and found that these reassortant viruses were fully competent in mammalian host systems. We also analyzed the currently available public data on the segment 8 gene distribution and found surprisingly little evidence for specific avian host restriction of the B-clade segment. We conclude that B-allele segment 8 genes are, in fact, capable of supporting infection in mammals and that they should be considered during the assessment of the pandemic risk of zoonotic influenza A viruses.

Please cite this paper as: Easterbrook et al. (2011) Obese mice have increased morbidity and mortality compared to non-obese mice during infection with the 2009 pandemic H1N1 influenza virus. Influenza and Other Respiratory Viruses 5(6), 418–425. Background Obesity has been identified as an independent risk factor for severe or fatal infection with 2009 pandemic H1N1 influenza (2009 pH1N1), but was not previously recognized for previous pandemic or seasonal influenza infections. Objectives Our aim was to evaluate the role of obesity as an independent risk factor for severity of infection with 2009 pH1N1, seasonal H1N1, or a pathogenic H1N1 influenza virus. Methods Diet-induced obese (DIO) and their non-obese, age-matched control counterparts were inoculated with a 2009 pH1N1, A/California/04/2009 (CA/09), current seasonal H1N1, A/NY/312/2001 (NY312), or highly pathogenic 1918-like H1N1, A/Iowa/Swine/1931 (Sw31), virus. Results Following inoculation with CA/09, DIO mice had higher mortality (80%) than control mice (0%) and lost more weight during infection. No effect of obesity on morbidity and mortality was observed during NY312 or Sw31 infection. Influenza antigen distribution in the alveolar regions of the lungs was more pronounced in DIO than control mice during CA/09 infection at 3 days post-inoculation (dpi), despite similar virus titers. During CA/09 infection, localized interferon-β and proinflammatory cytokine protein responses in the lungs were significantly lower in DIO than control mice. Conversely, serum cytokine concentrations were elevated in DIO, but not control mice following infection with CA/09. The effect of obesity on differential immune responses was abrogated during NY312 or Sw31 infection. Conclusions Together, these data support epidemiologic reports that obesity may be a risk factor for severe 2009 pandemic H1N1 influenza infection, but the role of obesity in seasonal or highly virulent pandemic influenza infection remains unclear.

The 1918 pandemic influenza virus has demonstrated significant pathogenicity in animal models and is the progenitor of ‘classical’ swine and modern seasonal human H1N1 lineages. Here we characterize the pathogenicity of an early ‘classical’ swine H1N1 influenza A virus isolated in 1931 compared to the pathogenicity of the 1918 pandemic virus and a seasonal H1N1 virus in mice and ferrets. A/Swine/Iowa/31 (Sw31) and the 1918 influenza viruses were uniformly lethal in mice at low doses and produced severe lung pathology. In ferrets, Sw31 and 1918 influenza viruses caused severe clinical disease and lung pathology with necrotizing bronchiolitis and alveolitis. The modern H1N1 virus caused little disease in either animal model. These findings revealed that in these models the virulence factors of the 1918 influenza virus are likely preserved in the Sw31 virus and suggest that early swine viruses may be a good surrogate model to study 1918 virulence and pathogenesis.

PA-X is a fusion protein of influenza A virus encoded in part from a +1 frameshifted X open reading frame (X-ORF) in segment 3. We show that the X-ORFs of diverse influenza A viruses can be divided into two groups that differ in selection pressure and likely function, reflected in the presence of an internal stop codon and a change in synonymous diversity. Notably, truncated forms of PA-X evolved convergently in swine and dogs, suggesting a strong species-specific effect.

Segment 7 of influenza A virus produces up to four mRNAs. Unspliced transcripts encode M1, spliced mRNA2 encodes the M2 ion channel, while protein products from spliced mRNAs 3 and 4 have not previously been identified. The M2 protein plays important roles in virus entry and assembly, and is a target for antiviral drugs and vaccination. Surprisingly, M2 is not essential for virus replication in a laboratory setting, although its loss attenuates the virus. To better understand how IAV might replicate without M2, we studied the reversion mechanism of an M2-null virus. Serial passage of a virus lacking the mRNA2 splice donor site identified a single nucleotide pseudoreverting mutation, which restored growth in cell culture and virulence in mice by upregulating mRNA4 synthesis rather than by reinstating mRNA2 production. We show that mRNA4 encodes a novel M2-related protein (designated M42) with an antigenically distinct ectodomain that can functionally replace M2 despite showing clear differences in intracellular localisation, being largely retained in the Golgi compartment. We also show that the expression of two distinct ion channel proteins is not unique to laboratory-adapted viruses but, most notably, was also a feature of the 1983 North American outbreak of H5N2 highly pathogenic avian influenza virus. In identifying a 14th influenza A polypeptide, our data reinforce the unexpectedly high coding capacity of the viral genome and have implications for virus evolution, as well as for understanding the role of M2 in the virus life cycle., Author Summary Influenza A virus is a pathogen capable of infecting a wide range of avian and mammalian hosts, causing seasonal epidemics and pandemics in humans. In recent years, the unexpected coding capacity of the virus has begun to be unravelled, with the identification of three more protein products (PB1-F2, PB1-N40 and PA-X) on top of the 10 viral proteins originally identified 30 years ago. Here, we identify a 14th primary translation product, made from segment 7. Previously established protein products from segment 7 include the matrix (M1) and ion channel (M2) proteins. M2, made from a spliced transcript, has multiple roles in the virus lifecycle including in entry and budding. In a laboratory setting, it is possible to generate M2 deficient viruses, but these are highly attenuated. However, upon serial passage a virus lacking the M2 splice donor site quickly recovered wild type growth properties, without reverting the original mutation. Instead we found a compensatory single nucleotide mutation had upregulated another segment 7 mRNA. This mRNA encoded a novel M2-like protein with a variant extracellular domain, which we called M42. M42 compensated for loss of M2 in tissue culture cells and animals, although it displayed some differences in subcellular localisation. Our study therefore identifies a further novel influenza protein and gives insights into the evolution of the virus.

Influenza's Cryptic Constraint Because of the well-known pandemic potential of influenza viruses, it is important to understand the range of molecular interactions between the virus and its host. Despite years of intensive research on the virus, Jagger et al. (p. 199 , published online 28 June; see the Perspective by Yewdell and Ince ) have found that the influenza A virus has been hiding a gene in its small negative-sense RNA genome. An overlapping open reading frame was found contained in the PA viral RNA polymerase gene, which is accessed by ribosomal frameshifting to produce a fusion protein containing the N-terminal messenger RNA (mRNA) endonuclease domain of PA and an alternative C-terminal X domain. The resulting polypeptide, PA-X, selectively degrades host mRNAs and, in a mouse model of infection, modulated cellular immune responses, thus limiting viral pathogenesis.

The 1918-1919 “Spanish” influenza pandemic is estimated to have caused 50 million deaths worldwide. Understanding the origin, virulence, and pathogenic properties of past pandemic influenza viruses, including the 1918 virus, is crucial for current public health preparedness and future pandemic planning. The origin of the 1918 pandemic virus has not been resolved, but its coding sequences are very like those of avian influenza virus. The proteins encoded by the 1918 virus differ from typical low-pathogenicity avian influenza viruses at only a small number of amino acids in each open reading frame. In this study, a series of chimeric 1918 influenza viruses were created in which each of the eight 1918 pandemic virus gene segments was replaced individually with the corresponding gene segment of a prototypical low-pathogenicity avian influenza (LPAI) H1N1 virus in order to investigate functional compatibility of the 1918 virus genome with gene segments from an LPAI virus and to identify gene segments and mutations important for mammalian adaptation. This set of eight “7:1” chimeric viruses was compared to the parental 1918 and LPAI H1N1 viruses in intranasally infected mice. Seven of the 1918 LPAI 7:1 chimeric viruses replicated and caused disease equivalent to the fully reconstructed 1918 virus. Only the chimeric 1918 virus containing the avian influenza PB2 gene segment was attenuated in mice. This attenuation could be corrected by the single E627K amino acid change, further confirming the importance of this change in mammalian adaptation and mouse pathogenicity. While the mechanisms of influenza virus host switch, and particularly mammalian host adaptation are still only partly understood, these data suggest that the 1918 virus, whatever its origin, is very similar to avian influenza virus.

Programmed ribosomal frameshifting is used in the expression of many virus genes and some cellular genes. In eukaryotic systems, the most well-characterized mechanism involves –1 tandem tRNA slippage on an X_XXY_YYZ motif. By contrast, the mechanisms involved in programmed +1 (or −2) slippage are more varied and often poorly characterized. Recently, a novel gene, PA-X, was discovered in influenza A virus and found to be expressed via a shift to the +1 reading frame. Here, we identify, by mass spectrometric analysis, both the site (UCC_UUU_CGU) and direction (+1) of the frameshifting that is involved in PA-X expression. Related sites are identified in other virus genes that have previously been proposed to be expressed via +1 frameshifting. As these viruses infect insects (chronic bee paralysis virus), plants (fijiviruses and amalgamaviruses) and vertebrates (influenza A virus), such motifs may form a new class of +1 frameshift-inducing sequences that are active in diverse eukaryotes.

Secondary bacterial infections increase disease severity of influenza virus infections and contribute greatly to increased morbidity and mortality during pandemics. To study secondary bacterial infection following influenza virus infection, mice were inoculated with sublethal doses of 2009 seasonal H1N1 virus (NIH50) or pandemic H1N1 virus (Mex09) followed by inoculation with Streptococcus pneumoniae 48 h later. Disease was characterized by assessment of weight loss and survival, titration of virus and bacteria by quantitative reverse transcription-PCR (qRT-PCR), histopathology, expression microarray, and immunohistochemistry. Mice inoculated with virus alone showed 100% survival for all groups. Mice inoculated with Mex09 plus S. pneumoniae showed severe weight loss and 100% mortality with severe alveolitis, denuded bronchiolar epithelium, and widespread expression of apoptosis marker cleaved caspase 3. In contrast, mice inoculated with NIH50 plus S. pneumoniae showed increased weight loss, 100% survival, and slightly enhanced lung pathology. Mex09-S. pneumoniae coinfection also resulted in increased S. pneumoniae replication in lung and bacteremia late in infection. Global gene expression profiling revealed that Mex09-S. pneumoniae coinfection did not induce significantly more severe inflammatory responses but featured significant loss of epithelial cell reproliferation and repair responses. Histopathological examination for cell proliferation marker MCM7 showed significant staining of airway epithelial cells in all groups except Mex09-S. pneumoniae-infected mice. This study demonstrates that secondary bacterial infection during 2009 H1N1 pandemic virus infection resulted in more severe disease and loss of lung repair responses than did seasonal influenza viral and bacterial coinfection. Moreover, this study provides novel insights into influenza virus and bacterial coinfection by showing correlation of lethal outcome with loss of airway basal epithelial cells and associated lung repair responses., IMPORTANCE Secondary bacterial pneumonias lead to increased disease severity and have resulted in a significant percentage of deaths during influenza pandemics. To understand the biological basis for the interaction of bacterial and viral infections, mice were infected with sublethal doses of 2009 seasonal H1N1 and pandemic H1N1 viruses followed by infection with Streptococcus pneumoniae 48 h later. Only infection with 2009 pandemic H1N1 virus and S. pneumoniae resulted in severe disease with a 100% fatality rate. Analysis of the host response to infection during lethal coinfection showed a significant loss of responses associated with lung repair that was not observed in any of the other experimental groups. This group of mice also showed enhanced bacterial replication in the lung. This study reveals that the extent of lung damage during viral infection influences the severity of secondary bacterial infections and may help explain some differences in mortality during influenza pandemics.

Pandemic influenza viral infections have been associated with viral pneumonia. Chimeric influenza viruses with the hemagglutinin segment of the 1918, 1957, 1968, or 2009 pandemic influenza viruses in the context of a seasonal H1N1 influenza genome were constructed to analyze the role of hemagglutinin (HA) in pathogenesis and cell tropism in a mouse model. We also explored whether there was an association between the ability of lung surfactant protein D (SP-D) to bind to the HA and the ability of the corresponding chimeric virus to infect bronchiolar and alveolar epithelial cells of the lower respiratory tract. Viruses expressing the hemagglutinin of pandemic viruses were associated with significant pathology in the lower respiratory tract, including acute inflammation, and showed low binding activity for SP-D. In contrast, the virus expressing the HA of a seasonal influenza strain induced only mild disease with little lung pathology in infected mice and exhibited strong in vitro binding to SP-D.

Influenza A virus segment 2 mRNA expresses three\ud polypeptides: PB1, PB1-F2 and PB1-N40, from AUGs\ud 1, 4 and 5 respectively. Two short open reading\ud frames (sORFs) initiated by AUGs 2 and 3 are also\ud present. To understand translational regulation in\ud this system, we systematically mutated AUGs 1–4\ud and monitored polypeptide synthesis from\ud plasmids and recombinant viruses. This identified\ud sORF2 as a key regulatory element with opposing\ud effects on PB1-F2 and PB1-N40 expression. We\ud propose a model in which AUGs 1–4 are accessed\ud by leaky ribosomal scanning, with sORF2 repressing\ud synthesis of downstream PB1-F2. However, sORF2\ud also up-regulates PB1-N40 expression, most likely\ud by a reinitiation mechanism that permits skipping of\ud AUG4. Surprisingly, we also found that in contrast to\ud plasmid-driven expression, viruses with improved\ud AUG1 initiation contexts produced less PB1 in\ud infected cells and replicated poorly, producing\ud virions with elevated particle:PFU ratios. Analysis\ud of the genome content of virus particles showed\ud reduced packaging of the mutant segment\ud 2 vRNAs. Overall, we conclude that segment\ud 2 mRNA translation is regulated by a combination\ud of leaky ribosomal scanning and reinitiation, and\ud that the sequences surrounding the PB1 AUG\ud codon are multifunctional, containing overlapping\ud signals for translation initiation and for segmentspecific\ud packaging.

Background. The overall impact of influenza virus infection in immunocompromised patients is largely unknown. Antigenic drift and genetic variations during prolonged influenza infection have been demonstrated. In this report we describe a multidrug-resistant H3N2 influenza virus isolated from an immunocompromised patient after 5 days of therapy. Methods. Multiple nasal wash samples were collected from an infected patient, and viral isolates were characterized. Sensitivity to antiviral agents was evaluated. Fitness and transmissibility were assessed in ferrets and tissue culture. Results. An in-frame 4―amino acid deletion emerged in the neuraminidase (NA) gene of an H3N2 virus after 5 days of oseltamivir therapy. No other changes in the NA or hemagglutinin genes were noted. Drug sensitivity assays revealed resistance to oseltamivir (>10-fold increase in 50% inhibitory concentration [IC 50 ]) and reduction in sensitivity to zanamivir (3-7-fold increase in IC 50 or 50% effective concentration). No change in fitness or transmissibility was observed. Conclusions. An in-frame NA gene deletion was rapidly selected for in an immunocompromised patient, resulting in decreased sensitivity of the isolate to available NA inhibitors without a change in fitness or transmissibility. This finding has implications for our understanding of the emergence of antiviral resistance and treatment of patients with influenza A infection, especially those who are immunocompromised.

Please cite this paper as: Kash et al. (2010) Prior infection with classical swine H1N1 influenza viruses is associated with protective immunity to the 2009 pandemic H1N1 virus. Influenza and Other Respiratory Viruses 4(3), 121–127. Background The 2009 H1N1 pandemic emerged even though seasonal H1N1 viruses have circulated for decades. Epidemio-logical evidence suggested that the current seasonal vaccine did not offer significant protection from the novel pandemic, and that people over the age of 50 might were less susceptible to infection. Objectives In a mouse challenge study with the 2009 pandemic H1N1 virus, we evaluated protective immune responses elicited by prior infection with human and swine influenza A viruses. Results Mice infected with A/Mexico/4108/2009 (Mex09) showed significant weight loss and 40% mortality. Prior infection with a 1976 classical swine H1N1 virus resulted in complete protection from Mex09 challenge. Prior infection with either a 2009 or a 1940 seasonal H1N1 influenza virus provided partial protection and a >100-fold reduction in viral lung titers at day 4 post-infection. Conclusions These findings indicate that in experimental animals recently induced immunity to 1918-derived H1N1 seasonal influenza viruses, and to a 1976 swine influenza virus, afford a degree of protection against the 2009 pandemic virus. Implications of these findings are discussed in the context of accumulating data suggesting partial protection of older persons during the 2009 pandemic.

Influenza A viruses are negative-strand segmented RNA viruses of the family Orthomyxoviridae that infect humans and a wide variety of animal species. Although there remains concern about the emergence of a pandemic influenza virus [1, 2], seasonal influenza epidemics consistently cause morbidity and mortality globally. Influenza viruses infect 5%–15% of the global population annually, resulting in ~500,000 deaths [3, 4]. In the United States, influenza is estimated to kill 36,000 people in an average year [5], which is significantly higher than the 15,000 deaths annually associated with AIDS [6]. Every few years, influenza epidemics boost annual mortality above average levels, causing 10,000–15,000 additional deaths [3, 4]. Influenza pandemics occur sporadically, characterized by a change in the viral hemagglutinin (HA) subtype through reassortment or introduction of a new virus (antigenic shift). Interpandemic influenza, in contrast, occurs yearly and has been characterized by acquisition of point mutations in the viral HA1 domain encoding the major antigenic sites of the HA protein, leading to serial antigenic change (antigenic drift) [7–9]. Recent studies have addressed the genomic evolutionary biology of interpandemic influenza [4, 10–13], suggesting that other viral features may play significant roles in strain selection and evolution. Co-circulation of multiple, genetically distinct strains leading to mixed infection and frequent intrasubtypic reassortment events [10, 12–14], as well as periodic “genome-wide selective sweeps’” characterized by the selection of a single clonal strain and the extinction of co-circulating strains [13], represent newly demonstrated evolutionary mechanisms, but biological correlations that lead to these phenomena are not well established. One important evolutionary event involved the emergence of the A/Fujian/411/2002-like (Fujian-like) viruses associated with the moderately severe epidemic of the 2003–2004 influenza season [16–18]. With diminished protection afforded by the influenza vaccine strains, pneumonia and influenza deaths in the United States during the 2003–2004 season exceeded the epidemic threshold for 9 consecutive weeks [19]. During the 2004–2005 A/California/7/2004-like (California-like) influenza season, the pneumonia and influenza deaths exceeded the epidemic threshold for 10 consecutive weeks [20]. During these seasons, the Centers for Disease Control and Prevention (CDC) reported an increase in deaths among children that was above the epidemic threshold [19–21]. Studies of Fujian-like viruses demonstrated the emergence of a major antigenic variant during the 2003–2004 influenza season that was derived from reassortment of 2 distinct clades of co-circulating H3N2 viruses, rather than by antigenic drift [10, 22]. These reassortant viruses contained the Fujian-like HA gene from a minor co-circulating H3N2 lineage (clade B) and the other 7 gene segments from the major circulating lineage (clade A) [10]. During the next influenza season, 2004–2005, another unexpected evolutionary event occurred. The clade A lineage that made up the majority of the H3N2 viruses during the 2003–2004 season was largely supplanted in 2004–2005 by the unreassorted clade B viruses, which acquired several HA antigenic changes to be become California-like [23, 24], but which also acquired a small number of amino acid mutations in the genes encoding the viral ribonucleoprotein complex (RNP). This derivative clade, termed here clade B′, caused a genome-wide selective sweep; that is, the replacement of the clade A viruses by the clade B′ viruses was so complete that the overall diversity of the circulating H3N2 viruses was dramatically reduced [12–14] (Figure 1). Figure 1 Genomic analyses of Fujian-like H3N2 viruses. A, Evolutionary relationships of concatenated major coding regions of influenza A viruses sampled in New York State during the period 1999–2004. The complete genomes of 35 human H3N2 influenza A virus ... In this study, we examined the hypothesis that Fujian-like viruses from the 2003–2004 influenza season (clades A and B) and the derivative California-like strain associated with the dominant selective sweep in 2004–2005 (clade B′) would exhibit different phenotypic properties that correlated with the selection of clade B′ viruses in 2004–2005 [13, 14]. The phenotypic properties of representative viruses from each clade were characterized in both cell culture and ferrets, and the role of the small number of amino acid mutations in the internal segments encoding the viral RNP of the clade B′ viruses was examined using an in vitro reporter assay.