Establishment Of Virus Infection Research Articles

A Noble Extract of Pseudomonas sp. M20A4R8 Efficiently Controlling the Influenza Virus-Induced Cell Death.

Epidemic diseases that arise from infectious RNA viruses, particularly influenza viruses, pose a constant threat to the global economy and public health. Viral evolution has undermined the efficacy of acquired immunity from vaccines and the antiviral effects of FDA-approved drugs. As such, there is an urgent need to develop new antiviral lead agents. Natural compounds, owing to their historical validation of application and safety, have become a promising solution. In this light, a novel marine bacterium, Pseudomonas sp. M20A4R8, has been found to exhibit significant antiviral activity [half maximal inhibitory concentration (IC50) = 1.3 µg/mL, selectivity index (SI) = 919.4] against influenza virus A/Puerto Rico/8/34, surpassing the activity of chloroquine. The antiviral response via M20A4R8 extract was induced during post-entry stages of the influenza virus, indicating suitability for post-application after the establishment of viral infection. Furthermore, post-treatment with M20A4R8 extract protected the host from virus-induced apoptosis, suggesting its potential use in acute respiratory disease complexes resulting from immune effectors' overstimulation and autophagy-mediated self-apoptosis. The extract demonstrated an outstanding therapeutic index against influenza virus A/Wisconsin/15/2009 (IC50 = 8.1 µg/mL, SI = 146.2) and B/Florida/78/2015 Victoria lineage (IC50 = 3.5 µg/mL, SI = 343.8), indicating a broad anti-influenza virus activity with guaranteed safety and effectiveness. This study provides a new perspective on mechanisms for preventing a broad spectrum of viral infections through antiviral agents from novel and natural origins. Future studies on a single or combined compound from the extract hold promise, encouraging its use in preclinical challenge tests with various influenza virus strains.

Host factors against plant viruses.

SummaryPlant virus genome replication and movement is dependent on host resources and factors. However, plants respond to virus infection through several mechanisms, such as autophagy, ubiquitination, mRNA decay and gene silencing, that target viral components. Viral factors work in synchrony with pro‐viral host factors during the infection cycle and are targeted by antiviral responses. Accordingly, establishment of virus infection is genetically determined by the availability of the pro‐viral factors necessary for genome replication and movement, and by the balance between plant defence and viral suppression of defence responses. Sequential requirement of pro‐viral factors and the antagonistic activity of antiviral factors suggest a two‐step model to explain plant–virus interactions. At each step of the infection process, host factors with antiviral activity have been identified. Here we review our current understanding of host factors with antiviral activity against plant viruses.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Crimean-Congo hemorrhagic fever virus nucleocapsid protein harbors distinct RNA-binding sites in the stalk and head domains

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus that causes severe hemorrhagic fever with a mortality rate of up to 30% in certain outbreaks worldwide. The virus has wide endemic distribution. There is no effective antiviral therapeutic or FDA approved vaccine for this zoonotic viral illness. The multifunctional CCHFV nucleocapsid protein (N protein) plays a crucial role in the establishment of viral infection and is an important structural component of the virion. Here we show that CCHFV N protein has a distant RNA-binding site in the stalk domain that specifically recognizes the vRNA panhandle, formed by the base pairing of complementary nucleotides at the 5' and 3' termini of the vRNA genome. Using multiple approaches, including filter-bonding analysis, GFP reporter assay, and biolayer interferometry we observed an N protein-panhandle interaction both in vitro and in vivo The purified WT CCHFV N protein and the stalk domain also recognize the vRNA panhandle of hazara virus, another Nairovirus in the family Bunyaviridae, demonstrating the genus-specific nature of N protein-panhandle interaction. Another RNA-binding site was identified at the head domain of CCHFV N protein that nonspecifically recognizes the single strand RNA (ssRNA) of viral or nonviral origin. Expression of CCHFV N protein stalk domain active in panhandle binding, dramatically inhibited the hazara virus replication in cell culture, illustrating the role of N protein-panhandle interaction in Nairovirus replication. Our findings reveal the stalk domain of N protein as a potential target in therapeutic interventions to manage CCHFV disease.

The Effects of High Temperature on Infection by Potato virus Y, Potato virus A, and Potato leafroll virus.

We examined the effects of temperature on acquisition of Potato virus Y-O (PVY-O), Potato virus A (PVA), and Potato leafroll virus (PLRV) by Myzus persicae by performing transmission tests with aphids that acquired each virus at different temperatures. Infection by PVY-O/PVA and PLRV increased with increasing plant temperature in Nicotiana benthamiana and Physalis floridana, respectively, after being transmitted by aphids that acquired them within a temperature range of 10–20°C. However, infection rates subsequently decreased. Direct qRT-PCR of RNA extracted from a single aphid showed that PLRV infection increased in the 10–20°C range, but this trend also declined shortly thereafter. We examined the effect of temperature on establishment of virus infection. The greatest number of plants became infected when N. benthamiana was held at 20°C after inoculation with PVY-O or PVA. The largest number of P. floridana plants became infected with PLRV when the plants were maintained at 25°C. PLRV levels were highest in P. floridana kept at 20–25°C. These results indicate that the optimum temperatures for proliferation of PVY-O/PVA and PLRV differed. Western blot analysis showed that accumulations of PVY-O and PVA coat proteins (CPs) were lower at 10°C or 15°C than at 20°C during early infection. However, accumulation increased over time. At 25°C or 30°C, the CPs of both viruses accumulated during early infection but disappeared as time passed. Our results suggest that symptom attenuation and reduction of PVY-O and PVA CP accumulation at higher temperatures appear to be attributable to increased RNA silencing.

Experimental pathological studies of an Indian chicken anaemia virus isolate and its detection by PCR and FAT.

Chicken Infectious Anaemia Virus (CIAV) is one of the potent immunosuppressive and economically important agents affecting poultry industry worldwide. Recent reports indicate the emergence of this virus in the poultry flocks of the country. The present study aimed to investigate the pathogenic potential of a recent isolate of CIAV obtained from poultry flock of Uttaranchal State, India. Twenty first day-old age Specific Pathogen Free (SPF) chicks were inoculated intramuscularly with 10(4.5) median tissue culture infective dose (TCID50) of CIAV passaged in the Marek's disease virus transformed chicken splenic T lymphocyte (MDCC-MSB 1) cell line while 15 chicks were kept as control. The CIAV isolate produced consistent clinical signs, loss in body weight gain, anaemia, low haematocrit values, bone marrow aplasia and generalized lymphoid atrophy. Mean Packed Cell Volume (PCV) value of the infected chicks was significantly low (18.22±2.22) compared to control group (34.12±4.72) at 14 day post infection (dpi). The establishment of virus infection in chicks was confirmed both at molecular and antigenic levels by Polymerase Chain Reaction (PCR) and Indirect Immunofluorescent Test (IIFT), respectively. Characteristic apoptotic pattern was also detected in the affected organs and the virus was re-isolated successfully in MDCC-MSB1 cell cultures. The present results revealed that the virus circulating in poultry flocks of Uttaranchal state is both pathogenic and immunosuppressive in nature. Extensive epidemiological studies are suggested in the poultry flocks of the country along with adaptation of appropriate diagnostic, prevention and control strategies so as to prevent economic losses caused by this important poultry pathogen.

End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3′X-tail region

The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA–RNA contacts important in the establishment of viral infection. Microarray antisense oligonucelotide assays, improved dimethyl sulfate probing methods and 2′ acylation chemistry (selective 2’-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3′ end to be regulated by the internal ribosome entry site (IRES) element via direct RNA–RNA interactions. The essential cis-acting replicating element (CRE) and the 3′X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5′ terminus. Further, the structural transition in the 3′X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3′X-tail region would therefore appear to occur. The preservation of this RNA–RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle.

Computational analysis predicts the Kaposi's sarcoma‐associated herpesvirus tegument protein ORF63 to be alpha helical

The innate immune response provides our first line of defence against infection. Over the course of evolution, pathogens have evolved numerous strategies to either avoid activating or to limit the effectiveness of the innate immune system. The Kaposi's sarcoma-associated herpesvirus (KSHV) contains tegument proteins in the virion that contribute to immune evasion and aid the establishment of viral infection. For example, the KSHV tegument protein ORF63 modulates inflammasome activation to inhibit the innate immune response against the virus. Understanding the likely structure of proteins involved in immune evasion enables potential mechanisms of action to be proposed. To understand more fully how ORF63 modulates the innate immune system we have utilized widely available bioinformatics tools to analyze the primary protein sequence of ORF63 and to predict its secondary and tertiary structure. We found that ORF63 is predicted to be almost entirely alpha-helical and may possess similarity to HEAT repeat containing proteins. Consequently, ORF63 is unlikely to be a viral homolog of the NLR protein family. ORF63 may inhibit the innate immune response by flexibly interacting with its target protein and inhibiting the recruitment of protein co-factors and/or conformational changes required for immune signaling.

Multitasking: Making the Most out of the Retroviral Envelope.

Evasion of the host’s immune system is a required step for the establishment of viral infection. In this article, we discuss the recent findings of Heidmann and colleagues demonstrating that some retroviruses possess an immune suppressive (IS) domain “encrypted” within their envelope glycoprotein that is required to establish a successful infection in immunocompetent hosts [1].

Hepatitis B virus X protein and simian virus 5 V protein exhibit similar UV-DDB1 binding properties to mediate distinct activities.

The UV-damaged DNA-binding activity protein (UV-DDB) consists of two subunits, DDB1 and DDB2, and functions in DNA repair and cell cycle regulation. The DDB1 subunit is a target for the hepatitis B virus X protein (HBx). Binding of HBx to DDB1 interferes with cell growth and viability in culture and has been implicated in the establishment of viral infection. DDB1 also interacts with the V proteins encoded by several paramyxoviruses including simian virus 5 (SV5), which prevent interferon signaling by targeting either STAT1 or STAT2 proteins for proteolysis. The role of V binding to DDB1, however, remains unclear. Here we show that the V protein of SV5 (SV5-V) and HBx exhibit strikingly similar DDB1 binding properties. Thus, SV5-V and HBx bind to DDB1 in a mutually exclusive manner, and SV5-V shares with HBx the ability to enhance the steady-state levels of DDB1 and to inhibit its association with DDB2. Yet only HBx induces cell death, and SV5-V can prevent HBx from doing so by blocking its interaction with DDB1. Binding of SV5-V to DDB1 may serve another function, since SV5-V shows a decreased ability to induce STAT1 degradation in cells expressing reduced amounts of DDB1. These findings demonstrate that HBx performs a unique function through its association with DDB1 for which SV5-V cannot substitute and suggest that SV5-V and HBx have evolved to bind DDB1 to achieve distinct functions, both by a mechanism that does not involve DDB2.

Nuclear localization of the duck hepatitis B virus capsid protein: detection and functional implications of distinct subnuclear bodies in a compartment associated with RNA synthesis and maturation.

We have reinvestigated the subcellular distribution of the duck hepatitis B virus (DHBV) core protein in infected duck hepatocytes and in transfected cells. By using indirect immunofluorescence, the protein was found to be localized not only in the cytoplasm, as described previously, but also within the cell nucleus, being concentrated in distinct, brightly staining nuclear core bodies (NCBs). In colocalization studies using confocal microscopy, the NCBs were found exclusively in the periphery of nuclear subdomains characterized as splicing factor compartments and distal to other subnuclear domains. Also relevant for their functional significance is that the NCBs formed during the establishment of virus infection, i.e., at very low overall concentrations of newly synthesized core protein, and persisted throughout all stages of infection. Moreover, a subset of NCBs colocalized with foci of pregenomic DHBV RNA present at concentrations detectable by fluorescence in situ hybridization. Taken together, these findings indicate that a minor fraction of the DHBV core protein molecules escapes the major cytoplasmic assembly pathway to accumulate in specific subnuclear domains, and they furthermore suggest that these NCBs serve a role in the synthesis and/or maturation of the DHBV RNA pregenome.

Turnover of hepatitis B virus X protein is regulated by damaged DNA-binding complex.

Mammalian hepatitis B viruses encode an essential regulatory protein, termed X, which may also be implicated in liver cancer development associated with chronic infection. X protein, also referred to as HBx in human virus and WHx in woodchuck virus, has been reported to bind to a number of cellular proteins, including the DDB1 subunit of the damaged DNA-binding (DDB) complex. Our previous work provided genetic evidence for the importance of WHx-DDB1 interaction in both the activity of the X protein and establishment of viral infection in woodchucks. In the present study, a direct action of DDB1 on the X protein is documented. Physical interaction between the two proteins leads to an increase in X protein stability. This effect results from protection of the viral protein from proteasome-mediated degradation. Protection of WHx is overcome in the presence DDB2, the second subunit of the DDB heterodimer. In keeping with observations reported for HBx, DDB2 was found to directly bind to WHx. Nonetheless, the counteracting effect of DDB2 on X stabilization requires DDB2-DDB1 interaction. Taken together, these findings substantiate the physical and functional connection between the X protein and the DDB1-DDB2 heterodimer, leading to the regulation of the pool of the viral protein.

An Infectious Clone of the West Nile Flavivirus

West Nile (WN) virus is the most widespread among flaviviruses, but until recently it was not known on the American continent. We describe here design of a subgenomic replicon, as well as a full-length infectious clone of the lineage II WN strain, which appeared surprisingly stable compared to other flavivirus infectious clones. This infectious clone was used to investigate effects of 5′- and 3′-nonrelated sequences on virus replication and infectivity of synthetic RNA. While a long nonrelated sequence at the 3′-end delayed but did not prevent establishment of the productive infectious cycle, a much shorter extra sequence at the 5′-end completely abrogated virus replication. Replacement of the conserved 5′-adenosine residue substantially delayed, but did not prevent, establishment of virus infection. In all cases, the recovered virus had restored its authentic 5′- and 3′-end genome sequences. However, the presence of extensive nonrelated sequences at both 5′- and 3′-ends could not be repaired.

Susceptibility to AZT of HIV-l Variants Grown in Epstein-Barr Virus-Transformed B Cell Lines

How 3'-azido-3'-deoxythymidine (AZT) affects the ability of three different variants of HIV-1 to infect a line of EBV-transformed B cells was studied. Susceptible cells were pretreated with three different concentrations of AZT (1, 5, and 10 microM) for 4 h before viral inoculation and were maintained after infection in drug-containing medium. Establishment of viral infection was assessed by indirect immunofluorescence for viral antigens and by antigen capture assay carried out on culture fluids. AZT completely blocked infection in one of the HIV-1 variants studied; in the other two, treatment of cells with AZT significantly delayed the appearance of progeny virus.

The infectibility of cowpea mesophyll cells by tobacco necrosis virus

The time required for infectious tobacco necrosis virus (TNV) to pass through the epidermis of cowpea leaves after mechanical inoculation, depended on plant age and environmental temperature. At 22°C the passage time was about 2 h but at 32°C it was only 10 min. Water stress seemed to play a role in the transport of infectious virus into the mesophyll. It was possible to infect cowpea mesophyll cells with TNV directly by using a fine brush without carborundum. Differences between tobacco and cowpea in the establishment of virus infection are discussed. De mogelijkheden om cowpea-mesofylcellen met TNV te infecteren, hetzij via de epidermis, hetzij rechtstreeks, werden onderzocht. De minimum tijd die infectieus virusmateriaal nodig heeft om de epidermis te passeren werd bepaald. De ouderdom van de zaailingen speelde daarbij een rol. Bij 20 dagen oude planten bedroeg de passagetijd meer dan 4 uur, in 10 dagen oude planten was meer dan 2 uur nodig voor passage. De temperatuur van de omgeving was een belangrijke factor. In cowpeabladeren, direct na inoculatie met TNV bij 32°C gezet, bleek de passgetijd slechts 10 minuten te zijn. Verduistering van de planten 24 uur voor inoculatie bekortte de passagetijd aanzienlijk. Ook speelde water-stress in het blad door hoge temperatuur na inoculatie een rol in de vroege infectiestadia. Het was mogelijk, cowpea-mesofyl direct te infecteren, indien cowpea-bladeren gebruikt werden van 9–12 dagen oude zaailingen die opgekweekt waren bij 22±3°C. De bladeren werden gedurende ±1 uur na afplukken op droog filtreerpapier gelegd, daarna werd de onderepidermis met een fijn pincet verwijderd. Inoculatie van het naakte mesofyl met TNV onmiddelljk na het strippen met een fijne penseel zonder carborundum resulteerde in eenzelfde aantal lesies als in de niet-gestripte helft, die geinoculeerd was door wrijven met hetzelfde TNV-inoculum in aanwezzigheid van carborundum. Het schijnt dat de processen, die betrokken zijn bij het tot stand komen van een TNV-infectie in cowpea en tabak, van elkaar verschillen.

Inhibition by ethidium bromide of the establishment of infection by murine sarcoma virus.

SUMMARY Using a non-cytotoxic dose of ethidium bromide, a differential inhibitor of mitochondrial DNA and RNA synthesis, some early event(s), required after infection of Balb 3T3 cells with murine sarcoma virus, did not occur. This led to an inhibition of virus replication. The cells became refractory to inhibition 24 h after infection. The most effective inhibition was observed when cells were treated with ethidium bromide for 15 to 18 h before infection, suggesting the depletion of a cellular material involved in the establishment of virus infection. The degree of inhibition by ethidium bromide appeared to be affected by the multiplicity of infection.

Effect of Ultra-Violet Radiation on the Respiration-Rates of Tobacco Leaves, and its Reversal by Visible Light

Bawden and Kleczkowski1 have shown that injury to the leaves of higher plants by exposure to ultra-violet light is prevented by subsequent exposure to visible light, and have related this effect to the phenomenon of ‘photo-reactivation’ or ‘photo-reversal’. During an investigation of the effect of irradiation on the establishment of virus infection in tobacco leaves evidence of a similar effect on leaf respiration has been obtained, and further investigation produced the results presented below.