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New therapies and vaccines based on nucleic acids combined with an efficient nanoparticle delivery vehicle have a broad applicability for different disease indications. An alternative delivery technology for the successfully applied lipid nanoparticles in mRNA SARS-CoV-2 vaccines are nanoparticles composed of biodegradable poly(amido)amine-based polymers with mRNA payload. To show that these polymeric nanoparticles can efficiently deliver influenza hemagglutinin mRNA to target tissues and elicit protective immune responses, a relevant ferret influenza challenge model was used. In this model, our nanoparticle-based vaccine elicited strong humoral and cellular immune responses in the absence of local and systemic reactogenicity. Upon virus challenge, vaccinated animals exhibited reduced clinical signs and virus load relative to unvaccinated control animals. Based on these findings, further investigation of the polymeric nanoparticles in the context of prophylactic vaccination is warranted. Future studies will focus on optimizing the payload, the nanoparticle stability, the efficacy in the context of pre-existing immunity, and the applicability of the technology to prevent other infectious diseases., Competing Interests: J.R., G.H., C.B., and R.G. are employees of 20Med Therapeutics., (© 2024 The Author(s).)

Animal models of influenza are important in preclinical research for the study of influenza infection and the assessment of vaccines, drugs and therapeutics. Here, we show that Golden Syrian hamsters ( Mesocricetus auratus ) inoculated via the intranasal route with high dose of influenza H1N1 display comparable disease kinetics and immune responses to the 'gold standard' ferret ( Mustela furo ) model. We demonstrate that both the hamster and ferret models have measurable disease endpoints of weight loss, temperature change, viral shedding from the upper respiratory tract and increased lung pathology. We also characterised both the humoral and cellular immune responses to infection in both models. The comparability of these data supports the Golden Syrian hamster model being useful in preclinical evaluation studies to explore the efficacy of countermeasures against influenza.

Cocirculation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses could pose unpredictable risks to health systems globally, with recent studies suggesting more severe disease outcomes in coinfected patients. The initial lack of a readily available coronavirus disease 2019 (COVID-19) vaccine has reinforced the importance of influenza vaccine programs during the COVID-19 pandemic. Live attenuated influenza vaccine (LAIV) is an important tool in protecting against influenza, particularly in children. However, it is unknown whether LAIV administration influences the outcomes of acute SARS-CoV-2 infection or disease. To investigate this, quadrivalent LAIV was administered to ferrets 3 days before or after SARS-CoV-2 infection. LAIV administration did not exacerbate the SARS-CoV-2 disease course or lung pathology with either regimen. In addition, LAIV administered before SARS-CoV-2 infection significantly reduced SARS-CoV-2 replication and shedding in the upper respiratory tract. This study demonstrated that LAIV administration in close proximity to SARS-CoV-2 infection does not exacerbate mild disease and can reduce SARS-CoV-2 shedding., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Live attenuated influenza vaccine (LAIV) is widely used to protect humans from seasonal influenza infection, particularly in children. In contrast to inactivated vaccines, the LAIV can induce both mucosal and cellular immune responses. Here we show that a single dose of monovalent H1N1pdm09-specific LAIV in the ferret model is fully protective against a subsequent wild-type H1N1pdm09 challenge, and furthermore reduces the severity of disease following challenge with a different influenza A subtype (H3N2). The reduced severity comprised reductions in weight loss and fever, as well as more rapid clearance of virus, compared to non-vaccinated H3N2-challenged ferrets. No H3N2-neutralizing antibodies were detected in vaccinated ferret sera. Rather, heterosubtypic protection correlated with interferon-gamma+ (IFN-γ+) T-cell responses measured in peripheral blood and in lung lymphocytes. The IFN-γ+ cells were cross-reactive to H3N2 virus even when obtained from vaccinated animals that had never been exposed to H3N2 virus. We believe this study provides compelling evidence that the LAIV can provide a significant reduction in infection and symptoms when challenged with heterosubtypic influenza strains not included in the LAIV, highlighting the importance of cross-reactive T-cells in the design of a universal influenza vaccine.

There is a vital need for authentic COVID-19 animal models to enable the pre-clinical evaluation of candidate vaccines and therapeutics. Here we report a dose titration study of SARS-CoV-2 in the ferret model. After a high (5 × 10 6  pfu) and medium (5 × 10 4  pfu) dose of virus is delivered, intranasally, viral RNA shedding in the upper respiratory tract (URT) is observed in 6/6 animals, however, only 1/6 ferrets show similar signs after low dose (5 × 10 2  pfu) challenge. Following sequential culls pathological signs of mild multifocal bronchopneumonia in approximately 5-15% of the lung is seen on day 3, in high and medium dosed groups. Ferrets re-challenged, after virus shedding ceased, are fully protected from acute lung pathology. The endpoints of URT viral RNA replication & distinct lung pathology are observed most consistently in the high dose group. This ferret model of SARS-CoV-2 infection presents a mild clinical disease.

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential for a diagnostic test for influenza virus which also provides insights on transmission, evolution, and drug resistance and simultaneously detects other viruses. We therefore set out to apply the Oxford Nanopore Technologies sequencing method to metagenomic sequencing of respiratory samples. We generated influenza virus reads down to a limit of detection of 10 2 to 10 3 genome copies/ml in pooled samples, observing a strong relationship between the viral titer and the proportion of influenza virus reads ( P  = 4.7 × 10 -5 ). Applying our methods to clinical throat swabs, we generated influenza virus reads for 27/27 samples with mid-to-high viral titers (cycle threshold [ C T ] values, <30) and 6/13 samples with low viral titers ( C T values, 30 to 40). No false-positive reads were generated from 10 influenza virus-negative samples. Thus, Nanopore sequencing operated with 83% sensitivity (95% confidence interval [CI], 67 to 93%) and 100% specificity (95% CI, 69 to 100%) compared to the current diagnostic standard. Coverage of full-length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza virus titers, we were able to reconstruct >99% complete sequences for all eight gene segments. We also detected a human coronavirus coinfection in one clinical sample. While further optimization is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza virus and other respiratory viruses., (Copyright © 2019 Lewandowski et al.)

Epidemiological data suggest that influenza vaccination protects against all-cause mortality in chronic obstructive pulmonary disease (COPD) patients. However, recent work has suggested there is a defect in the ability of some COPD patients to mount an adequate humoral response to influenza vaccination. The aim of our study was to investigate humoral and cell-mediated vaccine responses to the seasonal trivalent influenza vaccination (TIV) in COPD subjects and healthy controls. Forty-seven subjects were enrolled into the study; 23 COPD patients, 13 age-matched healthy controls (HC ≥ 50) and 11 young healthy control subjects (YC ≤ 40). Serum and peripheral blood mononuclear cells (PBMC) were isolated pre-TIV vaccination and at days 7 and 28 and 6 months post-vaccine for haemagglutinin inhibition (HAI) titre, antigen-specific T cell and antibody-secreting cell analysis. The kinetics of the vaccine response were similar between YC, HC and COPD patients and there was no significant difference in antibody titres between these groups at 28 days post-vaccine. As we observed no disease-dependent differences in either humoral or cellular responses, we investigated if there was any association of these measures with age. H1N1 (r = -0·4253, P = 0·0036) and influenza B (r = -0·344, P = 0·0192) antibody titre at 28 days negatively correlated with age, as did H1N1-specific CD4 + T helper cells (r = -0·4276, P = 0·0034). These results suggest that age is the primary determinant of response to trivalent vaccine and that COPD is not a driver of deficient responses per se. These data support the continued use of the yearly trivalent vaccine as an adjunct to COPD disease management., (© 2019 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

An effective universal vaccine for influenza will likely need to induce virus-specific T-cells, which are the major mediator of heterosubtypic cross-protection between different subtypes of influenza A virus. In this study we characterise the cell-mediated immune response in ferrets during heterosubtypic protection induced by low-dose H1N1 virus infection against an H3N2 virus challenge, given 4 weeks later. Although the ferrets were not protected against the infection by H3N2 virus, the duration of virus shedding was shortened, and clinical disease was markedly reduced. No cross-reactive neutralizing antibodies were detected, but cross-reactive interferon-gamma-secreting T cells were detected in the circulation prior to H3N2 challenge. These T-cells peaked at 11 days post-H1N1 infection, and were strongly induced in blood and in lung following H3N2 infection. The rapid induction of interferon-gamma-secreting cells in ferrets previously infected with H1N1 virus, but not in naïve ferrets, suggests induction of memory T-cells. These results are in accord with the observations that pre-existing cross-reactive T-cells correlate with protection in humans and have implications for outbreak modelling and universal vaccine design.

Seasonal influenza virus infections cause yearly epidemics which are the source of a significant public health burden worldwide. The ferret model for human influenza A virus (IAV) is widely used and has several advantages over other animal models such as comparable symptomology, similar receptor distribution in the respiratory tract to humans and the ability to be infected with human isolates without the need for adaptation. However, a major disadvantage of the model has been a paucity of reagents for the evaluation of the cellular immune response. Investigation of T-cell mediated immunity in ferrets is crucial to vaccine development and efficacy studies. In this study we have used commercially produced antibodies to ferret interferon gamma (IFN-γ) allowing us to reliably measure influenza-specific IFN-γ as a marker of the cellular immune response using both enzyme-linked immunospot (ELISpot) and enzyme-linked immunosorbent (ELISA) techniques. Here we demonstrate the application of these tools to evaluate cellular immunity in ferrets infected with clinically relevant seasonal H1N1 and H3N2 IAV subtypes at equivalent doses. Using small heparinised blood samples we were able to observe the longitudinal influenza-specific IFN-γ responses of ferrets infected with both seasonal subtypes of IAV and found a notable increase in influenza-specific IFN-γ responses in circulating peripheral blood within 8 days post-infection. Both seasonal strains caused a well-defined pattern of influenza-specific IFN-γ responses in infected ferrets when compared to naïve animals. Additionally, we found that while the influenza specific IFN-γ responses found in peripheral circulating blood were comparable between subtypes, the influenza specific IFN-γ responses found in lung lymphocytes significantly differed. Our results suggest that there is a distinct difference between the ability of the two seasonal influenza strains to establish an infection in the lung of ferrets associated with distinct signatures of acquired immunity., Competing Interests: The authors have declared that no competing interests exist.

Background: Defective interfering (DI) viruses are natural antivirals made by nearly all viruses. They have a highly deleted genome (thus being non-infectious) and interfere with the replication of genetically related infectious viruses. We have produced the first potential therapeutic DI virus for the clinic by cloning an influenza A DI RNA (1/244) which was derived naturally from genome segment 1. This is highly effective in vivo, and has unexpectedly broad-spectrum activity with two different modes of action: inhibiting influenza A viruses through RNA interference, and all other (interferon-sensitive) respiratory viruses through stimulating interferon type I., Results: We have investigated the RNA inhibitory mechanism(s) of DI 1/244 RNA. Ablation of initiation codons does not diminish interference showing that no protein product is required for protection. Further analysis indicated that 1/244 DI RNA interferes by replacing the cognate full-length segment 1 RNA in progeny virions, while interfering with the expression of genome segment 1, its cognate RNA, and genome RNAs 2 and 3, but not genome RNA 6, a representative of the non-polymerase genes., Conclusions: Our data contradict the dogma that a DI RNA only interferes with expression from its cognate full-length segment. There is reciprocity as cloned segment 2 and 3 DI RNAs inhibited expression of RNAs from a segment 1 target. These data demonstrate an unexpected complexity in the mechanism of interference by this cloned therapeutic DI RNA.

Background: Vaccination against seasonal influenza strains is recommended for "high risk" patient groups such as infants, elderly and those with respiratory or circulatory diseases. However, efficacy of the trivalent influenza vaccine (TIV) is poor in many cases and in the event of an influenza pandemic, mono-valent vaccines have been rapidly developed and deployed. One of the main issues with use of vaccine in pandemic situations is the lack of a suitable quantity of vaccine early enough during the pandemic to exert a major influence on the transmission of virus and disease outcome. One approach is to use a dose-sparing regimen which inevitably involves enhancing the efficacy using adjuvants., Methods: In this study we compare the use of a novel microcrystalline tyrosine (MCT) adjuvant, which is currently used in a niche area of allergy immunotherapy, for its ability to enhance the efficacy of a seasonal TIV preparation. The efficacy of the MCT adjuvant formulation was compared to alum adjuvanted TIV and to TIV administered without adjuvant using a ferret challenge model to determine vaccine efficacy., Results: The MCT was found to possess high protein-binding capacity. In the two groups where TIV was formulated with adjuvant, the immune response was found to be higher (as determined by HAI titre) than vaccine administered without adjuvant and especially so after challenge with a live influenza virus. Vaccinated animals exhibited lower viral loads (as determined using RT-PCR) than control animals where no vaccine was administered., Conclusions: The attributes of each adjuvant in stimulating single-dose protection against a poorly immunogenic vaccine was demonstrated. The properties of MCT that lead to the reported effectiveness warrants further exploration in this and other vaccine targets - particularly where appropriate immunogenic, biodegradable and stable alternative adjuvants are sought.

There is increasing evidence that 2009 pandemic H1N1 influenza viruses have evolved after pandemic onset giving rise to severe epidemics in subsequent waves. However, it still remains unclear which viral determinants might have contributed to disease severity after pandemic initiation. Here, we show that distinct mutations in the 2009 pandemic H1N1 virus genome have occurred with increased frequency after pandemic declaration. Among those, a mutation in the viral hemagglutinin was identified that increases 2009 pandemic H1N1 virus binding to human-like α2,6-linked sialic acids. Moreover, these mutations conferred increased viral replication in the respiratory tract and elevated respiratory droplet transmission between ferrets. Thus, our data show that 2009 H1N1 influenza viruses have evolved after pandemic onset giving rise to novel virus variants that enhance viral replicative fitness and respiratory droplet transmission in a mammalian animal model. These findings might help to improve surveillance efforts to assess the pandemic risk by emerging influenza viruses.

Currently, the need for cooled storage and the impossibility of terminal sterilisation are major drawbacks in vaccine manufacturing and distribution. To overcome current restrictions a preclinical safety and efficacy study was conducted to evaluate new influenza A vaccine formulations regarding thermal resistance, resistance against irradiation-mediated damage and storage stability. We evaluated the efficacy of novel antigen stabilizing and protecting solutions (SPS) to protect influenza A(H1N1)pdm09 split virus antigen under experimental conditions in vitro and in vivo. Original or SPS re-buffered vaccine (Pandemrix) was spray-dried and terminally sterilised by irradiation with 25 kGy (e-beam). Antigen integrity was monitored by SDS-PAGE, dynamic light scattering, size exclusion chromatography and functional haemagglutination assays. In vitro screening experiments revealed a number of highly stable compositions containing glycyrrhizinic acid (GA) and/or chitosan. The most stable composition was selected for storage tests and in vivo assessment of seroconversion in non-human primates (Macaca fascicularis) using a prime-boost strategy. Redispersed formulations with original adjuvant were administered intramuscularly. Storage data revealed high stability of protected vaccines at 4°C and 25°C, 60% relative humidity, for at least three months. Animals receiving original Pandemrix exhibited expected levels of seroconversion after 21 days (prime) and 48 days (boost) as assessed by haemagglutination inhibition and microneutralisation assays. Animals vaccinated with spray-dried and irradiated Pandemrix failed to exhibit seroconversion after 21 days whereas spray-dried and irradiated, SPS-protected vaccines elicited similar seroconversion levels to those vaccinated with original Pandemrix. Boost immunisation with SPS-protected vaccine resulted in a strong increase in seroconversion but had only minor effects in animals treated with non SPS-protected vaccine. In conclusion, utilising the SPS formulation technology, spray-drying and terminal sterilisation of influenza A(H1N1)pdm09 split virus vaccine is feasible. Findings indicate the potential utility of such formulated vaccines e.g. for needle-free vaccination routes and delivery to countries with uncertain cold chain facilities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Ferrets are widely used to study human influenza virus infection. Their airway physiology and cell receptor distribution makes them ideal for the analysis of pathogenesis and virus transmission, and for testing the efficacy of anti-influenza interventions and vaccines. The 2009 pandemic influenza virus (H1N1pdm09) induces mild to moderate respiratory disease in infected ferrets, following inoculation with 106 plaque-forming units (pfu) of virus. We have demonstrated that reducing the challenge dose to 102 pfu delays the onset of clinical signs by 1 day, and results in a modest reduction in clinical signs, and a less rapid nasal cavity innate immune response. There was also a delay in virus production in the upper respiratory tract, this was up to 9-fold greater and virus shedding was prolonged. Progression of infection to the lower respiratory tract was not noticeably delayed by the reduction in virus challenge. A dose of 104 pfu gave an infection that was intermediate between those of the 106 pfu and 102 pfu doses. To address the hypothesis that using a more authentic low challenge dose would facilitate a more sensitive model for antiviral efficacy, we used the well-known neuraminidase inhibitor, oseltamivir. Oseltamivir-treated and untreated ferrets were challenged with high (106 pfu) and low (102 pfu) doses of influenza H1N1pdm09 virus. The low dose treated ferrets showed significant delays in innate immune response and virus shedding, delayed onset of pathological changes in the nasal cavity, and reduced pathological changes and viral RNA load in the lung, relative to untreated ferrets. Importantly, these observations were not seen in treated animals when the high dose challenge was used. In summary, low dose challenge gives a disease that more closely parallels the disease parameters of human influenza infection, and provides an improved pre-clinical model for the assessment of influenza therapeutics, and potentially, influenza vaccines.

The emerging arbovirus Chandipura virus (CV) has been implicated in epidemics of acute encephalitis in India with high mortality rates. The isolation of temperature-dependent host-range (tdCE) mutants, which are impaired in growth at 39 °C in chick embryo (CE) cells but not in monkey cells, highlights a dependence on undetermined host factors. We have characterized three tdCE mutants, each containing one or more coding mutations in the RNA polymerase gene and two containing additional mutations in the attachment protein gene. Using reverse genetics, we showed that a single amino acid change in the virus polymerase of each mutant was responsible for the host-range specificity. In CE cells at the non-permissive temperature, the discrete cytoplasmic replication complexes seen in mammalian cells or at the permissive temperature in CE cells were absent with the tdCE mutants, consistent with the tdCE lesions causing disruption of the replication complexes in a host-dependent manner.

The main antivirals employed to combat seasonal and pandemic influenza are oseltamivir and zanamivir which act by inhibiting the virus-encoded neuraminidase. These have to be deployed close to the time of infection and antiviral resistance to the more widely used oseltamivir has arisen relatively rapidly. Defective interfering (DI) influenza virus is a natural antiviral that works in a different way to oseltamivir and zanamivir, and a cloned version (segment 1 244 DI RNA in a cloned A/PR/8/34 virus; 244/PR8) has proved effective in preclinical studies in mice. The active principle is the DI RNA, and this is thought to interact with all influenza A viruses by inhibiting RNA virus synthesis and packaging of the cognate virion RNA into nascent DI virus particles. We have compared the ability of DI virus and oseltamivir to protect ferrets from intranasal 2009 pandemic influenza virus A/California/04/09 (A/Cal, H1N1). Ferrets were treated with a single 2 μg intranasal dose of 244 DI RNA delivered as 244/PR8 virus, or a total of 25mg/kg body weight of oseltamivir given as 10 oral doses over 5 days. Both DI virus and oseltamivir reduced day 2 infectivity and the influx of cells into nasal fluids, and permitted the development of adaptive immunity. However DI virus, but not oseltamivir, significantly reduced weight loss, facilitated better weight gain, reduced respiratory disease, and reduced infectivity on days 4 and 6. 244 DI RNA was amplified by A/Cal by >25,000-fold, consistent with the amelioration of clinical disease. Treatment with DI virus did not delay clearance or cause persistence of infectious virus or DI RNA. Thus in this system DI virus was overall more effective than oseltamivir in combatting pandemic A/California/04/09., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Influenza A viruses are a major cause of morbidity and mortality in the human population, causing epidemics in the winter, and occasional worldwide pandemics. In addition there are periodic outbreaks in domestic poultry, horses, pigs, dogs, and cats. Infections of domestic birds can be fatal for the birds and their human contacts. Control in man operates through vaccines and antivirals, but both have their limitations. In the search for an alternative treatment we have focussed on defective interfering (DI) influenza A virus. Such a DI virus is superficially indistinguishable from a normal virus but has a large deletion in one of the eight RNAs that make up the viral genome. Antiviral activity resides in the deleted RNA. We have cloned one such highly active DI RNA derived from segment 1 (244 DI virus) and shown earlier that intranasal administration protects mice from lethal disease caused by a number of different influenza A viruses. A more cogent model of human influenza is the ferret. Here we found that intranasal treatment with a single dose of 2 or 0.2 µg 244 RNA delivered as A/PR/8/34 virus particles protected ferrets from disease caused by pandemic virus A/California/04/09 (A/Cal; H1N1). Specifically, 244 DI virus significantly reduced fever, weight loss, respiratory symptoms, and infectious load. 244 DI RNA, the active principle, was amplified in nasal washes following infection with A/Cal, consistent with its amelioration of clinical disease. Animals that were treated with 244 DI RNA cleared infectious and DI viruses without delay. Despite the attenuation of infection and disease by DI virus, ferrets formed high levels of A/Cal-specific serum haemagglutination-inhibiting antibodies and were solidly immune to rechallenge with A/Cal. Together with earlier data from mouse studies, we conclude that 244 DI virus is a highly effective antiviral with activity potentially against all influenza A subtypes.

We have shown earlier that a single dose of cloned defective interfering (DI) influenza A virus strongly protects mice from disease following a lethal challenge with different subtypes of influenza A virus. These animals suffered no clinical disease but experienced a subclinical infection which rendered them immune to reinfection with the same challenge virus. However, little is known about how DI virus achieves such protection. Here we investigated the role of adaptive immunity in DI virus-mediated protection using severe-combined immunodeficient (SCID) mice, which lack competence in both B- and T-cell compartments but retain NK cell activity. SCID mice which were treated with DI virus and infected with influenza virus initially remained completely well, while infected litter mates that received UV-inactivated DI virus became seriously ill and died. However, after 10 days of good health, the DI virus-protected SCID mice developed a clinical disease that was similar, but not completely identical, to the acute influenza disease. Disease was delayed longer by a higher dose of DI virus. We excluded the possibilities that the DI virus load in the lungs had declined, that the DI RNA sequence had changed so that it no longer interfered with the infectious genome, or that infectious virus had become resistant to the DI virus. These data show that while DI virus provides full protection from the acute disease in the absence of adaptive immunity, that same immunity is essential for clearing the infection. This indicates that the conventional view that DI virus-induced protection is mediated solely by competition for replication with the challenge virus is incorrect for influenza virus., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Chandipura virus (CV; genus Vesiculovirus, family Rhabdoviridae) is an emerging arbovirus, responsible for a number of outbreaks of severe viral encephalitis affecting children in India. A reverse genetics system has been constructed which allows recovery of infectious recombinant CV (rCV) entirely from cDNA. This system was used to construct a virus, rCVE, which has an additional transcription unit encoding green fluorescent protein (EGFP) between the 3rd and 4th CV genes. This virus grew to titres comparable to the parental rCV and stably expressed EGFP in infected cells for at least 4 passages. A second virus, rCVGM, was constructed in which the CV matrix (M) coding region was replaced with the coding region for an EGFP-M fusion protein. Compared to rCV and rCVE, rCVGM was attenuated, giving a small plaque phenotype and lower yield, although it did express the EGFP-M protein in infected cells. Passage of rCVGM resulted in viruses with a standard plaque phenotype which no longer expressed EGFP. Analysis of two of these viruses showed that most or all of the EGFP ORF was deleted. The EGFP-M fusion protein showed cleavage to EGFP-sized and M-sized products, both in rCVGM-infected cells and when expressed from a plasmid. The EGFP-M fusion protein was not detected in virus particles, suggesting it was incompatible with virus assembly, and particles of rCVGM likely contained the M-sized cleavage product in its place., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Influenza A and B viruses are major human respiratory pathogens that contribute to the burden of seasonal influenza. They are both members of the family Orthomyxoviridae but do not interact genetically and are classified in different genera. Defective interfering (DI) influenza viruses have a major deletion of one or more of their eight genome segments, which renders them both non-infectious and able to interfere in cell culture with the production of infectious progeny by a genetically compatible, homologous virus. It has been shown previously that intranasal administration of a cloned DI influenza A virus, 244/PR8, protects mice from various homologous influenza A virus subtypes and that it also protects mice from respiratory disease caused by a heterologous virus belonging to the family Paramyxoviridae. The mechanisms of action in vivo differ, with homologous and heterologous protection being mediated by probable genome competition and type I interferon (IFN), respectively. In the current study, it was shown that 244/PR8 also protects against disease caused by a heterologous influenza B virus (B/Lee/40). Protection from B/Lee/40 challenge was partially eliminated in mice that did not express a functional type I IFN receptor, suggesting that innate immunity, and type I IFN in particular, are important in mediating protection against this virus. It was concluded that 244/PR8 has the ability to protect in vivo against heterologous IFN-sensitive respiratory viruses, in addition to homologous influenza A viruses, and that it acts by fundamentally different mechanisms.

Background: We have identified and characterised a defective-interfering (DI) influenza A virus particles containing a highly deleted segment 1 RNA that has broad-spectrum antiviral activity. In young adult mice it exerts protection against several different subtypes of influenza A virus (defined here as homologous or genetically compatible protection) and against a paramyxovirus and an influenza B virus (heterologous or genetically unrelated protection). Homologous protection is mediated by replication competition between the deleted and full-length genomes, and heterologous protection occurs through stimulation of innate immunity, especially interferon type I., Methods: A single dose of the protective DI virus was administered intranasally to elderly mice at -7, -1 and +1 days relative to intranasal challenge with influenza A virus., Results: A single dose of the DI virus given 1 or 7 days protected elderly mice, reducing a severe, sometimes fatal disease to a subclinical or mild infection. In contrast, all members of control groups treated with inactivated DI virus before challenge became extremely ill and most died. Despite the subclinical/mild nature of their infection, protected mice developed solid immunity to a second infectious challenge., Conclusions: The defective interfering virus is effective in preventing severe influenza A in elderly mice and may offer a new approach to protection of the human population.

Respiratory viruses represent a major clinical burden. Few vaccines and antivirals are available, and the rapid appearance of resistant viruses is a cause for concern. We have developed a novel approach which exploits defective viruses (defective interfering (DI) or protecting viruses). These are naturally occurring deletion mutants which are replication-deficient and multiply only when coinfection with a genetically compatible infectious virus provides missing function(s) in trans. Interference/protection is believed to result primarily from genome competition and is therefore usually confined to the virus from which the DI genome originated. Using intranasally administered protecting influenza A virus we have successfully protected mice from lethal in vivo infection with influenza A viruses from several different subtypes [1]. Here we report, contrary to expectation, that protecting influenza A virus also protects in vivo against a genetically unrelated respiratory virus, pneumonia virus of mice, a pneumovirus from the family Paramyxoviridae. A single dose that contains 1μg of protecting virus protected against lethal infection. This protection is achieved by stimulating type I interferon and possibly other elements of innate immunity. Protecting virus thus has the potential to protect against all interferon-sensitive respiratory viruses and all influenza A viruses., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objectives: The cell surface receptor used by an influenza virus to infect that cell is an N-acetyl neuraminic acid (NANA) residue terminally linked by an alpha2,3 or alpha2,6 bond to a carbohydrate moiety of a glycoprotein or glycolipid. Our aim was to determine a quick and technically simple method to determine cell receptor usage by whole influenza A virus particles., Methods: We employed surface plasmon resonance to detect the binding of viruses to fetuin, a naturally occurring glycoprotein that has both alpha2,3- and alpha2,6-linked NANA, and free 3'-sialyllactose or 6'-sialyllactose to compete virus binding. All virus stocks were produced in embryonated chicken's eggs., Results: The influenza viruses tested bound preferentially to NANAalpha2,3Gal or to NANAalpha2,6Gal, or showed no preference. Two PR8 viruses had different binding preferences. Binding preferences of viruses correlated well with their known biological properties., Conclusions: Our data suggest that it is not easy to predict receptor usage by influenza viruses. However, direct experimental determination as described here can inform experiments concerned with viral pathogenesis, biology and structure. In principle, the methodology can be used for any virus that binds to a terminal NANA residue.

Defective interfering (DI) virus is simply defined as a spontaneously generated virus mutant from which a critical portion of the virus genome has been deleted. At least one essential gene of the virus is deleted, either in its entirety, or sufficiently to make it non-functional. The resulting DI genome is then defective for replication in the absence of the product(s) of the deleted gene(s), and its replication requires the presence of the complete functional virus genome to provide the missing functions. In addition to being defective DI virus suppresses production of the helper virus in co-infected cells, and this process of interference can readily be observed in cultured cells. In some cases, DI virus has been observed to attenuate disease in virus-infected animals. In this article, we review the properties of DI virus, potential mechanisms of interference and progress in using DI virus (in particular that derived from influenza A virus) as a novel type of antiviral agent.

Another influenza pandemic is inevitable, and new measures to combat this and seasonal influenza are urgently needed. Here we describe a new concept in antivirals based on a defined, naturally occurring defective influenza virus RNA that has the potential to protect against any influenza A virus in any animal host. This "protecting RNA" (244 RNA) is incorporated into virions which, although noninfectious, deliver the RNA to those cells of the respiratory tract that are naturally targeted by infectious influenza virus. A 120-ng intranasal dose of this 244 protecting virus completely protected mice against a simultaneous challenge of 10 50% lethal doses with influenza A/WSN (H1N1) virus. The 244 virus also protected mice against strong challenge doses of all other subtypes tested (i.e., H2N2, H3N2, and H3N8). This prophylactic activity was maintained in the animal for at least 1 week prior to challenge. The 244 virus was 10- to 100-fold more active than previously characterized defective influenza A viruses, and the protecting activity was confirmed to reside in the 244 RNA molecule by recovering a protecting virus entirely from cloned cDNA. There was a clear therapeutic benefit when the 244 virus was administered 24 to 48 h after a lethal challenge, an effect which has not been previously observed with any defective virus. Protecting virus reduced, but did not abolish, replication of challenge virus in mouse lungs during both prophylactic and therapeutic treatments. Protecting virus is a novel antiviral, having the potential to combat human influenza virus infections, particularly when the infecting strain is not known or is resistant to antiviral drugs.

Defective interfering (DI) virus RNAs result from major deletions in full-length viral RNAs that occur spontaneously during de novo RNA synthesis. These RNAs are packaged into virions that are by definition non-infectious, and are delivered to cells normally targeted by the virion. DI RNAs can only replicate with the aid of a coinfecting infectious helper virus, but the small size of DI RNA allows more copies of it to be made than of its full-length counterpart, so the cell produces defective virions in place of infectious progeny. In line with this scenario, the expected lethal disease in an influenza A virus-mouse model is made subclinical by administration of DI virus, but animals develop solid immunity to the infecting virus. Hence DI virus has been called an 'interfering vaccine'. Because interfering vaccine acts intracellularly and at a molecular level, it should be effective against all influenza A viruses regardless of subtype. Here we have used the ferret, widely acknowledged as the best model for human influenza. We show that an interfering vaccine with defective RNAs from an H3N8 virus almost completely abolished clinical disease caused by A/Sydney/5/97 (H3N2), with abrogation of fever and significant reductions in clinical signs of illness. Animals recovered fully and were solidly immune to reinfection, in line with the view that treatment converts the otherwise virulent disease into a subclinical and immunizing infection.

A defective interfering (DI) virus differs from the infectious virus from which it originated in having at least one major deletion in its genome. Such DI genomes are replicated only in cells infected in trans with homologous infectious virus and, as their name implies, they interfere with infectious virus replication and reduce the yield of progeny virus. This potent antiviral activity has been abundantly demonstrated in cell culture with many different DI animal viruses, but few in vivo examples have been reported, with the notable exception of DI Influenza A virus. A clue to this general lack of success arose recently when an anomaly was discovered in which DI Influenza A virus solidly protected mice from lethal disease caused by A/PR/8/34 (H1N1) and A/WSN/40 (H1N1) viruses, but protected only marginally from disease caused by A/Japan/305/57 (A/Jap, H2N2). The problem was not any incompatibility between the DI and infectious genomes, as A/Jap replicated the DI RNA in vivo. However, A/Jap required 300-fold more mouse infectious units to cause clinical disease than A/PR8 and it was hypothesized that it was this excess of infectivity that abrogated the protective activity of the DI virus. This conclusion was verified by varying the proportions of DI and challenge virus and showing that increasing the DI virus : infectious virus ratio in infected mice resulted in interference. Thus, counter-intuitively, DI virus is most effective against viruses that cause disease with low numbers of particles, i.e. virulent viruses.

Chandipura virus (CHPV) and Isfahan virus (ISFV) are two members of the genus Vesiculovirus from Asia. Both are arthropod-transmitted and are able to infect humans, but neither causes vesicular stomatitis in livestock. The complete genome sequence for each virus has been determined. The negative-sense RNA genome comprises 11,119 nt (CHPV) or 11,088 nt (ISFV). The most variable of the non-transcribed regions is the intergenic spacer at the G-L gene junction (4 bases in ISFV, 20 in CHPV). Phylogenetic analysis of deduced protein sequences shows that although CHPV and ISFV are distinct viruses, they are more related to each other than either is to the New World vesicular stomatitis viruses (VSV). The South American virus, Piry virus, is more closely related to the Asian viruses ISFV and CHPV, than it is to VSV.

The nucleocapsid (N) protein of the pneumovirus respiratory syncytial virus (RSV) is a major structural protein which encapsidates the RNA genome and is essential for replication and transcription of the RSV genome. The N protein of the related virus pneumonia virus of mice (PVM) is functionally unable to replace the RSV N protein in a minigenome replication assay. Using chimeric proteins, in which the immediate C-terminal part of the RSV N protein was replaced with the equivalent region of the PVM N protein, it was shown that six amino acid residues near the C terminus of the N protein (between residues 352-369) are essential for its function in replication and for the ability of the N protein to bind to the viral phosphoprotein, P.

The specificity of usage of promoters for replication and transcription by the pneumoviruses human respiratory syncytial virus (HRSV) and avian pneumovirus (APV) was studied using minigenomes containing a reporter gene. When infectious HRSV or APV was used as helper virus, replication could occur only if both the leader and trailer regions (containing the replicative and transcriptional promoters) were derived from the helper virus. In contrast, when the HRSV replication complex was supplied from cDNA plasmids, a minigenome containing either the APV leader or trailer was recognized and substantial levels of replication and transcription occurred. These data suggest that in pneumovirus-infected cells, helper virus functions can discriminate between genomes on the basis of the terminal sequences and that there is an association between the leader and trailer required for productive replication. This association is required only in virus-infected cells, not when replication and transcription are mediated by plasmid-directed expression of the component proteins required for replication and transcription. The possible implications of this are discussed.

The single amino acid change Gly172 to Ser in the phosphoprotein (P) of respiratory syncytial virus (RSV) has previously been shown to be responsible for the thermosensitivity and protein-negative phenotype of tsN19, a mutant of the B subgroup RSN-2 strain. This single change was inserted into the P gene of the A subgroup virus RSS-2, and the resulting phenotype was observed in a plasmid-driven reconstituted RSV RNA polymerase system. Expression from a genome analogue containing two reporter genes was thermosensitive when directed by plasmids containing the N, L, M2, and mutant P genes cloned under the control of T7 promoters. Analysis of RNA synthesis showed that mutant P protein was unable to produce genome, antigenome, or mRNA at the restrictive temperature. At a semipermissive temperature, genome, antigenome, and mRNA synthesis were all reduced, 6- to 30-fold, relative to synthesis directed by a wild-type P plasmid. Binding of the mutant P protein to N protein in the absence of other viral proteins was unaffected by temperature, indicating that the lesion did not produce a large enough structural change to disrupt this binding. These data suggest that the plasmid rescue system is suitable for investigation of the role of thermosensitive mutations in RSV polymerase components in RNA synthesis.

Chandipura virus (CHPV) is a Vesiculovirus, related to, but phylogenetically distinct from, vesicular stomatitis virus (VSV). The matrix protein of VSV, as well as its role in virus assembly, inhibits the transcription from promoters for host RNA polymerases I and II. Cloning and expression of the matrix protein of CHPV in human cells showed that this protein is also functional in its inhibitory effect on transcription of a reporter gene from the cytomegalovirus immediate-early promoter, despite sharing only 28% amino acid sequence identity with the matrix protein of VSV.

We have determined the nucleotide sequences of the regions 3' and 5' proximal to the avian pneumovirus (APV) N and L genes, respectively. These sequences were used in the construction of a synthetic minireplicon construct in which the chloramphenicol acetyltransferase (CAT) reporter gene was flanked at its 3' end with the APV leader together with the APV N gene start signal and at its 5' end with the APV L gene end signal and the genome trailer region. The ability of T7 RNA polymerase runoff transcripts to direct the replication and expression of the CAT reporter gene in APV-infected cells demonstrated the ability of the putative leader and trailer regions to direct genome replication and gene expression. Furthermore, this confirms the absence of the NS1 and NS2 gene analogs within the APV genome. We were able to detect the expression of CAT protein from cells that had been infected with supernatants from the initially infected and transfected cells. These results have identified the cis-acting sequences of APV responsible for viral replication, gene expression, and packaging into virus-like particles.

In Britain, grey squirrels (Sciurus carolinensis Gmelin) and pheasants (Phasianus colchicus Linnaeus) are important hosts of larvae and nymphs of Ixodes ricinus L., the principal European vector of the Lyme disease spirochaete, Borrelia burgdorferi sensu lato. To test whether squirrels are competent hosts of B. burgdorferi s.l., three females were trapped in the wild and then held in captivity. Following treatment, each animal was exposed to uninfected xenodiagnostic I. ricinus ticks. Squirrel A (an adult) which was inoculated experimentally with B. burgdorferi s.l., transmitted the infection to xenodiagnostic ticks. In contrast, squirrel B (a juvenile that was not inoculated)-showed no evidence of infection. Xenodiagnostic ticks that fed on control squirrel C (an adult) became infected and subsequently transmitted the infection experimentally to an uninfected hamster. The results indicated that squirrel C had a disseminated infection acquired in the wild and which persisted for at least 11 weeks. These data clearly demonstrate that grey squirrels are amplifying and reservoir hosts of B. burgdorferi s.l. The strain associated with squirrels was related to the B. afzelii genotype. Two observations implicated pheasants in a similar role: (i) a high prevalence of infection in engorged larvae collected from trapped pheasants, and (ii) the detection of B. burgdorferi s.l. (B. garinii genotype) in the wattle of 1/10 pheasants using PCR. Xenodiagnostic experiments similar to those undertaken with the squirrels are needed to confirm the role of pheasants in the transmission cycle of Lyme disease spirochaetes.

The nucleotide sequences of the genome of the RSS-2 wild type strain of respiratory syncytial (RS) virus, which is known to induce upper respiratory tract infection in adults, and that of the attenuated ts1C candidate vaccine derived from it by three cycles of mutagenesis and selection of temperature-sensitive (ts) mutants, have been determined. Comparison of the sequences has located the genetic changes which contribute to the reduced pathogenicity in adults of the candidate vaccine. Thirty-seven nucleotide changes distinguish the wild type and ts1C, 13 of which confer amino acid substitutions; no mutations are present in extragenic regions. Partial nucleotide sequencing of the genomes of the first stage ts mutant (ts1A) and the second stage ts mutant (ts1B), which were intermediates in the derivation of the third stage mutant ts1C, established that five mutations resulting in amino acid substitutions had been induced in the first cycle of mutagenesis, one in the second cycle, and seven in the third cycle. The unique mutation differentiating ts1B from ts1A substitutes an alanine for a threonine at residue 736 in the polymerase (L) protein. The occurrence of a mutation in ts1C inducing substitution of a phenylalanine for a serine residue at an adjacent site (731) suggests that mutations in this region of the polymerase can have significant attenuating effects. The data suggest also that a mutation in the F gene may contribute to the attenuated phenotype.

The nucleotide sequence of the large (L) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) was determined, completing the first entire genome sequence of a nairovirus. The L segment comprised 12255 nucleotides, making a total genome size of 18855 nucleotides, and the ends showed identity with the ends of the medium (M) and small (S) genomic segments. A single open reading frame (ORF) was present in the viral complementary strand, sufficient to encode a protein of 459 kDa. The predicted protein sequence showed the core polymerase motifs characteristic of the RNA-dependent RNA polymerases of segmented negative-stranded viruses. Comparison of the conserved motifs with the corresponding region of other segmented negative-strand viruses showed a closer relationship between nairoviruses and phleboviruses than with other Bunyaviridae or with other virus families. However, the core polymerase was the only function that could be assigned to a region of the DUG L gene.

The coding region of segment 4 of Thogoto (THO) virus, a tick-borne member of the Orthomyxoviridae, was expressed in a baculovirus system under the control of the polyhedrin promoter. This construct expressed authentic envelope glycoprotein as determined by size and antigenic reactivity with a panel of monoclonal antibodies (MAbs). Immunization of hamsters with Spodoptera frugiperda (Sf21) cells infected with the recombinant baculovirus induced neutralizing and protective antibodies against virus challenge; control hamsters developed clinical disease with high-titer viremia 3 days postchallenge. In contrast to hamsters, guinea pigs are comparatively resistant to THO virus infection but support nonviremic transmission between cofeeding infected and uninfected ticks. However, when uninfected ticks fed on guinea pigs immunized with the baculovirus recombinant, only 2% became infected following virus challenge of the animals compared to 26% of ticks on control nonimmune guinea pigs. Furthermore, neutralizing MAbs specific for THO viral glycoprotein protected mice against lethal challenge with THO virus; nonneutralizing MAbs specific for the glycoprotein, which inhibit THO viral agglutinating activity, did not induce a protective response. Thus at least in the murine model, protective immunity is conferred by antibodies directed against the neutralizing epitope(s) of the baculovirus-related glycoprotein of THO virus.

Diagnosis of Crimean-Congo haemorrhagic fever (CCHF) virus infections is hampered by the problems of handling this human pathogen, which requires the highest levels of biological containment. Recombinant antigens were examined for their potential as non-hazardous diagnostic reagents. The nucleocapsid (N) gene of the Greek AP92 isolate of CCHF virus was sequenced from cloned PCR products and the open reading frame was identified by homology to the N protein of a Chinese isolate of CCHF virus. The N protein was expressed to high levels in a baculovirus expression system. Three N protein-derived peptides were expressed in Escherichia coli as fusions with glutathione S-transferase and the antigenicities of these proteins and the baculovirus-expressed protein were tested by ELISA. When tested with laboratory animal sera representing all seven serogroups of nairoviruses, the only reactive sera were those raised to CCHF virus (Greek, Nigerian and Chinese isolates) and, more weakly, Hazara virus. When tested with a panel of known positive and negative human sera, the baculovirus-expressed N protein, and the peptide derived from the central region of the N protein, proved to be the best for identifying CCHF virus-specific IgG.

The coding assignments of the medium-sized (M) RNA segment of the Dugbe (DUG) virus (Nairovirus, Bunyaviridae) were investigated. The complete nucleotide sequence of 4888 nucleotides (nt) contained one long open reading frame in the viral complementary RNA, extending from an AUG start codon at nt 48-50 to a stop codon at nt 4701-4703 (numbered from the 5' terminus of vcRNA). Comparison of the terminal sequences with the ends of the DUG S segment revealed sequence identity between the first nine nucleotides of both segments. No sequence homologies were found with the M segments of other members of the Bunyaviridae, or with their polypeptide products. Expression of portions of the DUG M open reading frame in Escherichia coli demonstrated the carboxyl terminal region of the M open reading frame codes for the G1 structural glycoprotein, which is the target for neutralising antibodies. Confirmation of this assignment was obtained by sequencing the amino terminus of the G1 protein. Two nonstructural glycoproteins which share epitopes with G1 were identified in virus-infected cells, one of which (85 kDa) is processed over a period of several hours to produce G1. The G2 coding region was located upstream of the G1 sequence. The region between the carboxyl terminus of G2 and the 5' end of the long open reading frame apparently encodes a nonstructural protein of about 70 kDa, which is a precursor of the G2 protein.

The S RNA segments of the nairoviruses Crimean-Congo hemorrhagic fever (CCHF) virus (Chinese isolate) and Hazara (HAZ) virus were cloned and sequenced from PCR products. The RNAs comprise 1672 and 1677 nucleotides, respectively, and each encodes a protein in the viral complementary strand (54.0 and 54.2 kDa, respectively). The deduced protein sequences show homology to each other and to the nucleoprotein of Dugbe (DUG) nairovirus, although both the CCHF and HAZ viral proteins are larger. Alignment of the nucleoprotein sequences of CCHF, HAZ, and DUG viruses show that the CCHF and HAZ sequences are somewhat more closely related to each other (60.0% identity) than either is to the DUG sequence (55.4 and 53.0% identity, respectively); 39.5% of residues are identical across all three proteins. The carboxyl-terminus of DUG N protein shows a 40-residue deletion relative to the N proteins of the other two viruses.

The small (S) RNA segment of Dugbe (DUG) virus (Nairovirus, Bunyaviridae) encodes a single protein, the nucleocapsid (N) protein, of M(r) 49.4 kDa. cDNA derived from the complete coding region for the N protein was cloned into Autographa californica nuclear polyhedrosis virus (AcNPV) under control of the polyhedrin promoter and used to infect Spodoptera frugiperda insect cells. Western blotting analysis using monoclonal antibodies demonstrated the production of DUG N protein in the infected cells. Monoclonal and polyclonal antibodies to the N protein of Crimean-Congo haemorrhagic fever (CCHF) virus were found to cross-react weakly with the baculovirus expressed DUG N protein by Western blotting. When used in an enzyme linked immunoassay (ELISA), the DUG N protein reacted with polyclonal mouse immune ascitic fluids raised against either CCHF or Hazara viruses (both members of the CCHF serogroup of nairoviruses). Cross-reactions between DUG virus (Nairobi sheep disease serogroup) and members of other nairovirus serogroups were not detected.

Thogoto (THO) virus is a tick-transmitted virus which shares morphological and biochemical characteristics with members of the Orthomyxoviridae. The genome of Thogoto virus comprises six segments of single-stranded, negative sense RNA. The complete nucleotide sequence of the fourth largest RNA segment of THO virus has been determined from cDNA analyses. This RNA segment is 1574 nt long and has a coding capacity for a glycoprotein of 512 amino acids with a predicted molecular weight of 57,550 Da. The sequence of this protein has extensive homology with the putative envelope protein of Dhori (DHO) virus, the only other recorded tick-borne orthomyxo-like virus, but not with the envelope glycoproteins of the influenza viruses. A search of the translation of the available nucleotide database has produced evidence for a relationship between the glycoproteins of THO and DHO viruses and the gp64 glycoprotein of two DNA-containing insect baculoviruses, Autographa californica nuclear polyhedrosis virus and Orgyia pseudotsugata nuclear polyhedrosis virus. The baculovirus gp64 protein is a membrane protein implicated in endocytotic fusion events during infection. A multiple alignment between these four glycoproteins gave significance scores of greater than 28 standard deviations, indicating that the homologies between them are highly significant. The distribution of cysteine residues is conserved between all four proteins which also have similar hydropathy profiles, suggestive of a type I membrane protein topology.

The dissemination and replication of Dugbe (DUG) virus and its tissue tropisms in the tick vector Amblyomma variegatum were examined by immunohistochemical analysis using specific antibody, in situ hybridization with a viral-complementary riboprobe, and infectivity assays of dissected tissues. Dugbe virus was localized in both unfed and feeding adults inoculated as nymphs or orally infected by capillary feeding, and in nymphs infected by capillary feeding. In non-feeding ticks, the main sites of DUG virus replication were the epidermis, hemocytes associated with loose connective tissue, and a small number of phagocytic digestive cells in the gut lumen. Virus infectivity in the hemolymph was associated entirely with hemocytes. Dugbe viral antigen or infectivity was not detected in the salivary glands until after the start of feeding. Viral titers in the salivary glands of feeding ticks were about ten-fold higher than in gut, ovary, or loose connective tissue. The level of infection decreased during molting and increased during feeding. Viral particles and pathologic effects were not detected in infected ticks. The primary site of trans-stadial persistence of DUG virus is the hemocytes. Tick hemocytes and other motile cells may be important in the transmission of persistent virus infection from one cell or organ to another by diapedesis.

The complete nucleotide sequence (1448 nucleotides) of RNA 2 of a Czechoslovakian isolate TpM-34 of red clover necrotic mosaic virus (RCNMV-TpM-34) has been determined. The sequence contained one major open reading frame (ORF) with the potential to encode a protein of 326 amino acids (Mr 35755), designated P2. The nucleotide sequence of RNA 2 of RCNMV-TpM-34 and the previously published sequence of RNA 2 of an Australian isolate of the virus (RCNMV-Aus) were 83% identical and there was 80% amino acid sequence identity between the P2 proteins of these isolates. However the N-terminal two-thirds of the P2 proteins shared a higher degree of similarity than the C-terminal regions which were predicted to have a more flexible structure. An ORF in the 3' portion of RNA 2 of RCNMV-Aus, which could encode a protein of Mr 5000, was not present in RNA 2 of RCNMV-TpM-34. RNAs 1 and 2 of RCNMV-TpM-34 and RCNMV-Aus are bilaterally compatible.

The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection.

Cloned cDNA derived from the small (S) and medium (M) genomic RNA segments of Dugbe (DUG) virus, isolate ArD44313, a member of the Nairobi sheep disease (NSD) serogroup of nairoviruses (family, Bunyaviridae) was used to prepare 32P-labelled DNA and RNA probes. The S and M segments of six isolates of DUG virus all hybridised to both DNA and RNA probes, although the M segment of isolate KT281/75 reacted only weakly. Of nine other nairoviruses tested, representing all the six other serogroups within the Nairovirus genus, none hybridised to the DNA probes. However, under conditions of low stringency, the DUG S and M RNA probes hybridised to the respective S and M segments of Ganjam (GAN) virus (another member of the NSD serogroup). The DUG S RNA probe also hybridised to the S segments of Crimean-Congo haemorrhagic fever (CCHF) virus and Hazara (HAZ) virus (members of the CCHF serogroup). The indicated sequence relationships between DUG, GAN, CCHF and HAZ viruses show that the NSD serogroup is more closely related to members of the CCHF serogroup than it is to nairoviruses of the other five serogroups.

The S RNA segment of Dugbe (DUG) virus (Nairovirus; Bunyaviridae) was sequenced from three overlapping cDNA clones and by primer extension. The S RNA is 1712 nucleotides in length and contains one large open reading frame (ORF) of 1326 nucleotides coding for a 49.4-kDa protein on viral complementary (vc) RNA. This protein in size corresponds to the DUG nucleocapsid (N) protein (P. Cash, 1985, J. Gen. Virol. 66, 141-148). The 49.4-kDa product was expressed as a fusion protein with beta-galactosidase in Escherichia coli cells and confirmed as DUG N protein by Western blotting with DUG N-specific monoclonal antibody. An additional ORF of 150 nucleotides coding for a possible 5.9-kDa protein is present in the +1 reading frame, 3' to the N protein ORF on vcRNA. DUG S segment mRNA was found to be essentially full length. No evidence was obtained for the existence of a smaller mRNA species that could code for a 5.9-kDa protein. Comparisons of the DUG S RNA sequence and predicted N protein amino acid sequence, with the respective sequences of snowshoe hare, La Crosse (bunyaviruses), Punta Toro, Sandfly fever Sicilian (phleboviruses), and Hantaan (hantavirus) viruses, failed to detect any sequence similarity, although the genomic structure of DUG S RNA is similar to that of the S RNA segment of Hantaan (HTN) virus.

The complete nucleotide sequence of the S RNA segment of Sandfly Fever Sicilian (SFS) virus (Phlebovirus, Bunyaviridae) was determined from overlapping cDNA clones and by primer extension. The RNA is 1746 nucleotides in length and has two large open reading frames (ORF), one of which (24.8 kDa) is viral-complementary in sense, and the other (30.4 kDa) is in the viral sense. This ambisense genome arrangement has been seen in another member of the Phlebovirus genus, Punta Toro (PT) virus (T. Ihara, H. Akashi, and D. H. L. Bishop, 1984, Virology 136, 293-306), but not in representatives of either the Bunyavirus or Hantavirus genera of the Bunyaviridae. Comparison of the predicted amino acid sequences for SFS virus with the recognized products of PT S RNA (T. Ihara, Y. Matsuura, and D. H. L. Bishop, 1985, Virology 147, 317-325; H. A. Overton, T. Ihara, and D. H. L. Bishop, 1987, Virology 157, 338-350) indicated that the 24.8-kDa ORF encodes the nucleoprotein (N) of SFS virus, and the 30.4-kDa ORF codes for a nonstructural protein (NSs). Subgenomic messenger RNAs, from which these two proteins are presumably translated, were detected in virus-infected cells.