Metagenomic Analysis of the Ferret Fecal Viral Flora

Ferrets are widely used as a small animal model for a number of viral infections, including influenza A virus and SARS coronavirus. To further analyze the microbiological status of ferrets, their fecal viral flora was studied using a metagenomics approach. Novel viruses from the families Picorna-, Papilloma-, and Anelloviridae as well as known viruses from the families Astro-, Corona-, Parvo-, and Hepeviridae were identified in different ferret cohorts. Ferret kobu- and hepatitis E virus were mainly present in human household ferrets, whereas coronaviruses were found both in household as well as farm ferrets. Our studies illuminate the viral diversity found in ferrets and provide tools to prescreen for newly identified viruses that potentially could influence disease outcome of experimental virus infections in ferrets.     

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

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The symptom difference induced by <Emphasis Type="Italic">Tobacco mosaic virus</Emphasis> and <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> in tobacco plants containing the <Emphasis Type="Italic">N</Emphasis> gene is determined by movement protein gene

Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) are two closely related viruses in the genus Tobamovirus, but they induce obviously different sizes of necrotic lesions in tobacco plants containing the N gene. Comparison of the symptoms produced by TMV, ToMV and a chimaeric virus (T/OMP), in which the TMV movement protein (MP) gene was replaced by the ToMV MP gene, showed T/OMP caused necrotic lesions that were similar in size to those of ToMV in tobacco plants containing the N gene. The coat protein and MP of the three viruses accumulated in planta with similar levels, and the replication level of TMV and T/OMP in protoplasts also had no difference. Comparison of the activities of defense-related enzymes (PAL, POD and PPO) induced by the three viruses also showed that the variability of enzyme activity induced by T/OMP was similar to that induced by TMV, but different from that induced by ToMV. The results indicate that the size difference of necrotic lesions induced by TMV and ToMV in tobacco plants containing the N gene results from the functional difference of their MP genes.     

Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses,Paramyxoviruses, Filoviruses,and Influenza Viruses

Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed.     

Knockout of SlTOM1 and SlTOM3 results in differential resistance to tobamovirus in tomato

During tobamovirus–host coevolution, tobamoviruses developed numerous interactions with host susceptibility factors and exploited these interactions for replication and movement. The plant‐encoded TOBAMOVIRUS MULTIPLICATION (TOM) susceptibility proteins interact with the tobamovirus replicase proteins and allow the formation of the viral replication complex. Here CRISPR/Cas9‐mediated mutagenesis allowed the exploration of the roles of SlTOM1a, SlTOM1b, and SlTOM3 in systemic tobamovirus infection of tomato. Knockouts of both SlTOM1a and SlTOM3 in sltom1a/sltom3 plants resulted in an asymptomatic response to the infection with recently emerged tomato brown rugose fruit virus (ToBRFV). In addition, an accumulation of ToBRFV RNA and coat protein (CP) in sltom1a/sltom3 mutant plants was 516‐ and 25‐fold lower, respectively, than in wild‐type (WT) plants at 12 days postinoculation. In marked contrast, sltom1a/sltom3 plants were susceptible to previously known tomato viruses, tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV), indicating that SlTOM1a and SlTOM3 are not essential for systemic infection of TMV and ToMV in tomato plants. Knockout of SlTOM1b alone did not contribute to ToBRFV and ToMV resistance. However, in triple mutants sltom1a/sltom3/sltom1b, ToMV accumulation was three‐fold lower than in WT plants, with no reduction in symptoms. These results indicate that SlTOM1a and SlTOM3 are essential for the replication of ToBRFV, but not for ToMV and TMV, which are associated with additional susceptibility proteins. Additionally, we showed that SlTOM1a and SlTOM3 positively regulate the tobamovirus susceptibility gene SlARL8a3. Moreover, we found that the SlTOM family is involved in the regulation of plant development.     

Recombinant viral sialate-O-acetylesterases

Viral O-acetylesterases were first identified in several viruses, including influenza C viruses and coronaviruses. These enzymes are capable of removing cellular receptors from the surface of target cells. Hence they are also known as receptor destroying enzymes. We have cloned and expressed several recombinant viral O-acetylesterases. These enzymes were secreted from Sf9 insect cells as chimeric proteins fused to eGFP. A purification scheme to isolate the recombinant O-acetylesterase of influenza C virus was developed. The recombinant enzymes derived from influenza C viruses specifically hydrolyze 9-O-acetylated sialic acids, while that of sialodacryoadenitis virus, a rat coronavirus related to mouse hepatitis virus, is specific for 4-O-acetylated sialic acid. The recombinant esterases were shown to specifically de-O-acetylate sialic acids on glycoconjugates. We have also expressed esterase knockout proteins of the influenza C virus hemagglutinin-esterase. The recombinant viral proteins can be used to unambiguously identify O-acetylated acids in a variety of assays. Published in 2004..     

Down on the farm: Farm coronaviruses are a large reservoir for new spillover events into humans

Until COVID‐19, coronaviruses were largely overlooked by virologists. Yet, their abundance in mammalian species could cause new spillover events into humans. Subject Categories: Ecology, Microbiology, Virology & Host Pathogen Interaction

By now, coronaviruses have become notorious. Yet, before the SARS coronaviruses started wreaking havoc with human society, coronaviruses were largely overlooked, even amongst virologists. “When I told another virologist at a meeting what I was working on”, recalled Peter Rottier at Utrecht University, the Netherlands, who began his coronavirus research in 1979, “that person said they''d never heard of coronaviruses”. With plenty of other viruses such as influenza and HIV demanding attention, funders saw no reason to support research on the endemic human coronaviruses in the 1980s and 1990s since they caused mostly common cold.

With plenty of other viruses such as influenza and HIV demanding attention, funders saw no reason to support research on the endemic human coronaviruses in the 1980s and 90s…

Clinicians and virologists began taking much notice around 2003 and 2004 after the SARS coronavirus inflicted about 800 deaths before the outbreak receded, but the potential of this virus family to spillover into new species and mutate to more virulent strains had long been recognized by veterinarians, because coronaviruses inflict significant diseases in poultry, cattle and especially pigs. Virologists now are taking a closer look at some of these diseases to better understand coronaviruses and why they may be relevant for potential future pandemics.     

A Live Attenuated H7N7 Candidate Vaccine Virus Induces Neutralizing Antibody That Confers Protection from Challenge in Mice,Ferrets, and Monkeys

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of highly pathogenic (HP) A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs). Intranasal (i.n.) administration of a single dose of the H7N7 NL/03 ca vaccine virus fully protected mice from lethal challenge with homologous and heterologous H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03 ca vaccine induced neutralizing antibodies in serum and provided complete protection from pulmonary replication of homologous and heterologous wild-type H7 challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine elicited an antibody response in one of three AGMs that was completely protected from pulmonary replication of the homologous wild-type H7 challenge virus. The contribution of CD8⁺ and/or CD4⁺ T cells to the vaccine-induced protection of mice was evaluated by T-cell depletion; T lymphocytes were not essential for the vaccine-induced protection from lethal challenge with H7 wt viruses. Additionally, passively transferred neutralizing antibody induced by the H7N7 NL/03 ca virus protected mice from lethality following challenge with H7 wt viruses. The safety, immunogenicity, and efficacy of the H7N7 NL/03 ca vaccine virus in mice, ferrets, and AGMs support the evaluation of this vaccine virus in phase I clinical trials.Highly pathogenic avian influenza (HPAI) is a disease of poultry that is caused by H5 or H7 avian influenza viruses and is associated with up to 100% mortality (2). Influenza A H7 subtype viruses from both Eurasian and North American lineages have resulted in more than 100 cases of human infection since 2002 in the Netherlands, Italy, Canada, the United Kingdom, and the United States. These cases include outbreaks of HPAI H7N7 virus in the Netherlands in 2003 that resulted in more than 80 cases of human infection and one fatality; HPAI H7N3 virus in British Columbia, Canada, in 2004 that resulted in two cases of conjunctivitis; a cluster of human infections of low-pathogenicity avian influenza (LPAI) H7N2 virus in the United Kingdom in 2007 that resulted in several cases of influenza-like illness and conjunctivitis; and a single case of respiratory infection in New York in 2003 (3-6, 17, 27).Due to an unprecedented geographic spread of H5 subtype viruses since 2003 and the continued occurrence of sporadic cases of H5N1 infections in humans, much emphasis has been placed on the pandemic threat posed by H5 subtype viruses. However, H7 subtype viruses also have significant pandemic potential. Humans are immunologically naïve to the H7 avian influenza viruses (16), and LPAI H7 subtype viruses circulating in domestic poultry and wild birds in Eurasia and North America have the potential to evolve and acquire an HP phenotype either by accumulating mutations or by recombination at the hemagglutinin (HA) cleavage site resulting in a highly cleavable HA that is a virulence motif in poultry (30, 33, 34). Recent work also suggests that contemporary North American lineage H7 subtype viruses, isolated in 2002 to 2003, are partially adapted to recognize α2-6-linked sialic acids, which are the receptors preferred by human influenza viruses and are preferentially found in the human upper respiratory tract (7). Moreover, coinfection and genetic reassortment of RNA genomes between H7 avian influenza viruses and human influenza viruses, including the seasonal H1N1 and H3N2 and pandemic H1N1 viruses, could result in the generation of reassortant viruses with the capacity to efficiently transmit among people and result in a pandemic. Domesticated birds may serve as important intermediate hosts for the transmission of wild-bird influenza viruses to humans, as may pigs, as evidenced by human infections with swine-origin 2009 pandemic H1N1 influenza virus throughout the world.Vaccination is the most effective method for the prevention of influenza. However, technical limitations result in delays in the rapid generation and availability of a strain-specific vaccine against an emerging pandemic virus. The emergence of antigenically distinct virus clades poses a substantial challenge for the design of vaccines against H5N1 viruses because of the possible need for clade-specific vaccines (1). Similar challenges are present for the generation of H7 subtype vaccine candidates, because antigenically distinct H7 subtype viruses, including North American lineage H7N2 and H7N3 and Eurasian lineage H7N7 and H7N3 viruses, have caused human disease. The successful control of H7 influenza virus in poultry has been achieved by stamping out and by vaccination of poultry (9). Vaccines for human use against both lineages of H7 influenza virus are under development, and candidate vaccines have been evaluated in preclinical and clinical studies (14, 23, 29, 42).We have previously analyzed the antigenic relatedness among H7 viruses from Eurasian and North American lineages using postinfection mouse and ferret sera (22). Among 10 H7 viruses tested, A/Netherlands/219/03 (H7N7) virus induced the most broadly cross-neutralizing antibodies (Abs) (22). Based on the phylogenetic relationships and its ability to induce broadly cross-neutralizing antibodies in mice and ferrets, we selected the A/Netherlands/219/03 (NL/03) (H7N7) virus from the Eurasian lineage for vaccine development. We used reverse genetics to generate a live attenuated cold-adapted (ca) H7N7 candidate vaccine virus bearing a modified HA, a wild-type (wt) neuraminidase (NA) gene from the NL/03 wt virus, and the six internal protein gene segments from the cold-adapted (ca) influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (AA ca) (H2N2). The immunogenicity and protective efficacy against challenge with HP and LP H7 viruses from the Eurasian and North American lineages of the reassortant H7N7 NL03/AA ca vaccine virus were evaluated in mice, ferrets, and African green monkeys (AGMs).     

Interferon-λ Contributes to Innate Immunity of Mice against Influenza A Virus but Not against Hepatotropic Viruses

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-α, IFN-β and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-λ uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1^0/0) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-λ might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-λ readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1^0/0 mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-λ failed to induce Mx1 in the liver of IFNAR1^0/0 mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-λ receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-α/β and IFN-λ were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1^0/0 mice. From these results we conclude that IFN-λ contributes to inborn resistance against viral pathogens infecting the lung but not the liver.     

芽胞杆菌及其代谢物抑制包膜病毒感染的研究进展

包膜病毒指具有一层脂质双层膜的病毒,如流感病毒、冠状病毒等,这些包膜病毒每年在世界范围内导致许多严重的疾病,严重威胁人类健康。使用抗病毒药物是预防与治疗病毒感染的主要策略,芽胞杆菌(Bacillus)及其代谢物能够抑制多种包膜病毒的感染。本文综述了芽胞杆菌代谢的粗提物、肽、酶、胞外聚合物、小双链RNA和热灭活的枯草芽胞杆菌孢子在抗包膜病毒感染中发挥的重要作用,其机制是通过直接破坏病毒包膜、阻止膜融合、与病毒基因组RNA直接配对、催化裂解病毒RNA、激活天然免疫反应等对抗病毒,期望为包膜病毒的持续预防和治疗提供参考。     

Evaluation of live attenuated influenza a virus h6 vaccines in mice and ferrets

Avian influenza A virus A/teal/HK/W312/97 (H6N1) possesses seven gene segments that are highly homologous to those of highly pathogenic human influenza H5N1 viruses, suggesting that a W312-like H6N1 virus might have been involved in the generation of the A/HK/97 H5N1 viruses. The continuous circulation and reassortment of influenza H6 subtype viruses in birds highlight the need to develop an H6 vaccine to prevent potential influenza pandemics caused by the H6 viruses. Based on the serum antibody cross-reactivity data obtained from 14 different H6 viruses from Eurasian and North American lineages, A/duck/HK/182/77, A/teal/HK/W312/97, and A/mallard/Alberta/89/85 were selected to produce live attenuated H6 candidate vaccines. Each of the H6 vaccine strains is a 6:2 reassortant ca virus containing HA and NA gene segments from an H6 virus and the six internal gene segments from cold-adapted A/Ann Arbor/6/60 (AA ca), the master donor virus that is used to make live attenuated influenza virus FluMist (intranasal) vaccine. All three H6 vaccine candidates exhibited phenotypic properties of temperature sensitivity (ts), ca, and attenuation (att) conferred by the internal gene segments from AA ca. Intranasal administration of a single dose of the three H6 ca vaccine viruses induced neutralizing antibodies in mice and ferrets and fully protected mice and ferrets from homologous wild-type (wt) virus challenge. Among the three H6 vaccine candidates, the A/teal/HK/W312/97 ca virus provided the broadest cross-protection against challenge with three antigenically distinct H6 wt viruses. These data support the rationale for further evaluating the A/teal/HK/W312/97 ca vaccine in humans.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.     

Detection of Infectious Tobamoviruses in Forest Soils

Our objectives were to evaluate elution and bait plant methods to detect infectious tobamoviruses in forest soils in New York State. Soils were collected from two forest sites: Whiteface Mountain (WF) and Heiberg Forest (HF). The effectiveness of four buffers to elute tomato mosaic tobamovirus (ToMV) from organic and mineral fractions of WF soil amended with ToMV was tested, and virus content was assessed by enzyme-linked immunosorbent assay (ELISA). The effectiveness of Chenopodium quinoa (Willd.) bait plants to detect the virus also was tested. Both methods then were utilized to detect tobamoviruses in 11 WF and 2 HF soil samples. A phosphate buffer (100 mM, pH 7.0) eluted more ToMV from soil than the other buffers tested. Mineral soil bound more virus than organic soil. Virus recoveries from virus-amended organic and mineral soils were 3 and 10%, respectively, and the detection sensitivity was 10 to 20 ng/g of soil. Roots of bait plants grown in all virus-amended soils tested positive by ELISA, and virus concentrations averaged 10 ng/g. Both ToMV and tobacco mosaic tobamovirus (TMV) were transmitted to C. quinoa by elution from one of two HF soil samples but not from the WF soil samples. A tobamovirus was detected by bait planting in 12 of 73 (16%) root extracts representing 5 of 13 soil samples (38%). Tobamovirus-like particles were seen by transmission electron microscopy in 6 of 12 infected root extracts. Tobamoviruses occur in forest soils in New York State. Abiotic soil transmission to trees may permit localized spread and persistence of these viruses in forest ecosystems.     

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses.     

A novel genetic locus linked to pro-inflammatory cytokines after virulent H5N1 virus infection in mice

Background

Genetic variation in the human population is a key determinant of influenza disease severity. A single nucleotide polymorphism in the antiviral gene IFITM3 was linked to outcomes during the 2009 H1N1 pandemic. To identify variant host genes associated with increased virus replication and severe disease, we performed a quantitative trait locus analysis on pro-inflammatory cytokine production 48 hours after intranasal infection with highly pathogenic H5N1 influenza virus.

Results

Pro-inflammatory cytokines CCL2, TNFα and IFN-α, were measured by ELISA in lung homogenates of DBA/2J (D2), C57BL/6J (B6) and 44 different BXD recombinant inbred mouse strains. Virus titer was also assessed in a subset of these animals. CCL2 (8-fold), TNFα (24-fold) and IFN-α (8-fold) concentrations varied significantly among the different BXD RI strains. Importantly, cytokine concentration correlated very well (r =0.86-0.96, P <0.0001) with virus titer suggesting that early cytokine production is due to increased virus infection and replication. Linkage analysis of cytokine concentration revealed a significant locus on chromosome 6 associated with differences in TNFα, IFN-α and CCL2 cytokine concentration (LRS =26). This locus accounted for nearly 20% of the observed phenotypic variation in the BXD population studied. Sequence and RNA expression analysis identified several candidate host genes containing missense mutations or deletions; Samd9l, Ica1, and Slc25a13. To study the role of Slc25a13, we obtained Slc25a13 knockout line, but upon challenge with H5N1 influenza virus observed no effect on CCL2 production, or morbidity and mortality.

Conclusion

A novel genetic locus on chromosome 6 modulates early pro-inflammatory cytokine production and virus replication after highly pathogenic influenza virus infection. Candidate genes, Samd9l and Ica1, may be important for the control of influenza virus infection and pathogenesis.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1017) contains supplementary material, which is available to authorized users.     

Avian Influenza Virus Glycoproteins Restrict Virus Replication and Spread through Human Airway Epithelium at Temperatures of the Proximal Airways

Transmission of avian influenza viruses from bird to human is a rare event even though avian influenza viruses infect the ciliated epithelium of human airways in vitro and ex vivo. Using an in vitro model of human ciliated airway epithelium (HAE), we demonstrate that while human and avian influenza viruses efficiently infect at temperatures of the human distal airways (37°C), avian, but not human, influenza viruses are restricted for infection at the cooler temperatures of the human proximal airways (32°C). These data support the hypothesis that avian influenza viruses, ordinarily adapted to the temperature of the avian enteric tract (40°C), rarely infect humans, in part due to differences in host airway regional temperatures. Previously, a critical residue at position 627 in the avian influenza virus polymerase subunit, PB2, was identified as conferring temperature-dependency in mammalian cells. Here, we use reverse genetics to show that avianization of residue 627 attenuates a human virus, but does not account for the different infection between 32°C and 37°C. To determine the mechanism of temperature restriction of avian influenza viruses in HAE at 32°C, we generated recombinant human influenza viruses in either the A/Victoria/3/75 (H3N2) or A/PR/8/34 (H1N1) genetic background that contained avian or avian-like glycoproteins. Two of these viruses, A/Victoria/3/75 with L226Q and S228G mutations in hemagglutinin (HA) and neuraminidase (NA) from A/Chick/Italy/1347/99 and A/PR/8/34 containing the H7 and N1 from A/Chick/Italy/1347/99, exhibited temperature restriction approaching that of wholly avian influenza viruses. These data suggest that influenza viruses bearing avian or avian-like surface glycoproteins have a reduced capacity to establish productive infection at the temperature of the human proximal airways. This temperature restriction may limit zoonotic transmission of avian influenza viruses and suggests that adaptation of avian influenza viruses to efficient infection at 32°C may represent a critical evolutionary step enabling human-to-human transmission.     

Coincident ruddy turnstone migration and horseshoe crab spawning creates an ecological ‘hot spot’ for influenza viruses

Since 1985, avian influenza virus surveillance has been conducted annually from mid-May to early June in charadriiform species from the families Scolopacidae and Laridae (shorebirds and gulls) at Delaware Bay in the northeast United States. The mass migrations of shorebirds, gulls and horseshoe crabs (Limulus polyphemus) coincide at that time, and large numbers of migrating birds pause at Delaware Bay to feed on horseshoe crab eggs deposited at the high-tide line. Influenza viruses are consistently isolated from charadriiform birds at Delaware Bay, at an overall rate approximately 17 times the combined rate of isolation at all other surveillance sites worldwide (490 isolates/9474 samples, 5.2% versus 49 isolates per 15 848 samples, 0.3%, respectively; Proportion test, p < 0.0001). The likelihood of isolating influenza viruses at Delaware Bay is dependent on the presence of ruddy turnstone (Arenaria interpres) at the sampling site (G-test of independence, p < 0.001). The convergence of host factors and environmental factors results in a unique ecological ‘hot spot’ for influenza viruses in Charadriiformes.     

Insertion of a Multibasic Cleavage Motif into the Hemagglutinin of a Low-Pathogenic Avian Influenza H6N1 Virus Induces a Highly Pathogenic Phenotype

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR↓G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA₀ (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.Wild birds represent the natural reservoir of avian influenza A viruses in nature (43). Influenza A viruses are classified on the basis of the hemagglutinin (HA) and neuraminidase (NA) surface glycoproteins. In wild birds throughout the world, influenza A viruses representing 16 HA and 9 NA antigenic subtypes have been found in numerous combinations (also called subtypes, e.g., H1N1, H6N1) (12). Besides classification based on the antigenic properties of HA and NA, avian influenza A viruses can also be classified based on their pathogenic phenotype in chickens. Highly pathogenic avian influenza (HPAI) virus, an acute generalized disease of poultry in which mortality may be as high as 100%, is restricted to subtypes H5 and H7. Other avian influenza A virus subtypes are generally low-pathogenic avian influenza (LPAI) viruses that cause much milder, primarily respiratory disease in poultry, sometimes with loss of egg production (6).The HA protein of influenza A virus is initially synthesized as a single polypeptide precursor (HA₀), which is cleaved into HA₁ and HA₂ subunits by host cell proteases. The mature HA protein mediates binding of the virus to host cells, followed by endocytosis and HA-mediated fusion with endosomal membranes (43). Influenza viruses of subtypes H5 and H7 may become highly pathogenic after introduction into poultry and cause outbreaks of HPAI. The switch from an LPAI phenotype to the HPAI phenotype of these H5 and H7 influenza A viruses is achieved by the introduction of basic amino acid residues into the HA₀ cleavage site by substitution or insertion, resulting in the so-called multibasic cleavage site (MBCS), which facilitates systemic virus replication (4, 5, 14, 44). The cleavage of the HA₀ of LPAI viruses is restricted to trypsin-like proteases which recognize the XXX(R/K)↓G cleavage motif, where the downward arrow indicates the site of cleavage. Replication of these LPAI viruses is therefore restricted to sites in the host where these enzymes are expressed, i.e., the respiratory and intestinal tract (32, 38). The introduction of an RX(R/K)R↓G or R(R/K)XR↓G minimal MBCS motif into the H5 and H7 subtype viruses facilitates the recognition and cleavage of the HA₀ by ubiquitous proprotein convertases, such as furin (20, 32, 41, 45). H5 influenza A viruses with a minimal MBCS motif only have the highly pathogenic phenotype if the masking glycosylation site at position 11 in the HA is replaced by a nonglycosylation site. Otherwise, at least one additional basic amino acid has to be inserted to allow the shift from an LPAI virus phenotype to an HPAI virus phenotype to occur (15, 18, 21, 22, 28). No information is available on the minimal prerequisites of H7 influenza A viruses to become highly pathogenic, but all HPAI H7 viruses have at least 2 basic amino acid insertions in the HA₀ cleavage site (22). HA₀ with the MBCS is activated in a broad range of different host cells and therefore enables HPAI viruses to replicate systemically in poultry (46). To date, little is known about the apparent subtype-specific nature of the introduction of the MBCS into LPAI viruses and the evolutionary processes involved in the emergence of HPAI viruses. When an MBCS was introduced in a laboratory-adapted strain of influenza virus, A/Duck/Ukraine/1/1963 (H3N8), it did not result in a dramatic change in pathogenic phenotype (35). Here, the effect of the introduction of an MBCS into a primary LPAI H6N1 virus, A/Mallard/Sweden/81/2002, is described. The introduction of an MBCS resulted in trypsin-independent replication in vitro and enhanced pathogenesis in a chicken model. Understanding the basis of the HA subtype specificity of the introduction of an MBCS into avian influenza viruses will lead to a better understanding of potential molecular restrictions involved in emergence of HPAI outbreaks.     

Beyond malaria: The inhibition of viruses by artemisinin-type compounds

Natural products represent valuable chemical scaffolds for drug development. A recent success story in this context was artemisinin, which is not only active against malaria but also to other diseases. This raised the interest of artemisinin's potential for drug repurposing. On the present review, we give an overview on artemisinin's antiviral activity. There is good in vitro and in vivo evidence for the activity of artemisinin and its derivatives against DNA viruses of the Herpesviridae and Hepadnaviridae families such as cytomegaloviruses, human herpesvirus 6, herpes simplex viruses 1 and 2, Epstein-Barr virus and Hepatitis B virus. The evidence is weaker for Polyomaviruses and papilloma viruses. Weaker or no inhibitory activity in vitro has been reported for RNA viruses such as human immunodeficiency viruses 1 and 2, hepatitis C virus, influenza virus and others. Interestingly, the artemisinin derivative artesunate did not exert cross-resistance to ganciclovir-resistant HCMV and exerted synergistic inhibition in combination with several clinically established antiviral standard drugs. The antiviral activity of first generation artemisinin derivatives (e.g. artesunate, artemether, etc.) was enhanced by novel derivatives, including dimer and trimer molecules. First results on patients indicating activity in a subset of HCMV patients. Novel developments in the field of nanotechnology and synthetic biology to bioengineer microorganisms for artemisinin production may pave the way for novel drugs to fight viral infections with artemisinin-based drugs.