Reassortment between Avian H5N1 and Human Influenza Viruses Is Mainly Restricted to the Matrix and Neuraminidase Gene Segments

Highly pathogenic avian influenza H5N1 viruses have devastated the poultry industry in many countries of the eastern hemisphere. Occasionally H5N1 viruses cross the species barrier and infect humans, sometimes with a severe clinical outcome. When this happens, there is a chance of reassortment between H5N1 and human influenza viruses. To assess the potential of H5N1 viruses to reassort with contemporary human influenza viruses (H1N1, H3N2 and pandemic H1N1), we used an in vitro selection method to generate reassortant viruses, that contained the H5 hemagglutinin gene, and that have a replication advantage in vitro. We found that the neuraminidase and matrix gene segments of human influenza viruses were preferentially selected by H5 viruses. However, these H5 reassortant viruses did not show a marked increase in replication in MDCK cells and human bronchial epithelial cells. In ferrets, inoculation with a mixture of H5N1-pandemic H1N1 reassortant viruses resulted in outgrowth of reassortant H5 viruses that had incorporated the neuraminidase and matrix gene segment of pandemic 2009 H1N1. This virus was not transmitted via aerosols or respiratory droplets to naïve recipient ferrets. Altogether, these data emphasize the potential of avian H5N1 viruses to reassort with contemporary human influenza viruses. The neuraminidase and matrix gene segments of human influenza viruses showed the highest genetic compatibility with HPAI H5N1 virus

Influenza A viruses are transmitted via the air from the nasal respiratory epithelium of ferrets

Human influenza A viruses are known to be transmitted via the air from person to person. It is unknown from which anatomical site of the respiratory tract influenza A virus transmission occurs. Here, pairs of genetically tagged and untagged influenza A/H1N1, A/H3N2 and A/H5N1 viruses that are transmissible via the air are used to co-infect donor ferrets via the intranasal and intratracheal routes to cause an upper and lower respiratory tract infection, respectively. In all transmission cases, we observe that the viruses in the recipient ferrets are of the same genotype as the viruses inoculated intranasally, demonstrating that they are expelled from the upper respiratory tract of ferrets rather than from trachea or the lower airways. Moreover, influenza A viruses that are transmissible via the air preferentially infect ferret and human nasal respiratory epithelium. These results indicate that virus replication in the upper respiratory tract, the nasal respiratory epithelium in particular, of donors is a driver for transmission of influenza A viruses via the air

A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication

RNA structures are increasingly recognized to be of importance during influenza A virus replication. Here, we investigated a predicted conserved hairpin in the M gene segment (nt 967-994) within the region of the vRNA 5′ packaging signal. The existence of this RNA structure and its possible role in virus replication was investigated using a compensatory mutagenesis approach. Mutations were introduced in the hairpin stem, based on natural variation. Virus replication properties were studied for the mutant viruses with disrupted and restored RNA structures. Viruses with structure-disrupting mutations had lower virus titers and a significantly reduced median plaque size when compared with the wild-type (WT) virus, while viruses with structure restoring-mutations replicated comparable to WT. Moreover, virus replication was also reduced when mutations were introduced in the hairpin loop, suggesting its involvement in RNA interactions. Northern blot and FACS experiments were performed to study differences in RNA levels as well as production of M1 and M2 proteins, expressed via alternative splicing. Stem-disruptive mutants caused lower vRNA and M2 mRNA levels and reduced M2 protein production at early time-points. When the RNA structure was restored, vRNA, M2 mRNA and M2 protein levels were increased, demonstrating a compensatory effect. Thus, this study provides evidence for functional importance of the predicted M RNA structure and suggests its role in splicing regulation

Middle east respiratory syndrome coronavirus (MERS-CoV) serology in major livestock species in an affected region in Jordan, june to September 2013

Between June and September 2013, sera from 11 dromedary camels, 150 goats, 126 sheep and 91 cows were collected in Jordan, wher

Possible increased pathogenicity of Pandemic (H1N1) 2009 Influenza virus upon reassortment

Since emergence of the pandemic (H1N1) 2009 virus in April 2009, three influenza A viruses-seasonal (H3N2), seasonal (H1N1), and pandemic (H1N1) 2009-have circulated in humans. Genetic reassortment between these viruses could result in enhanced pathogenicity. We compared 4 reassortant viruses with favorable in vitro replication properties with the wild-type pandemic (H1N1) 2009 virus with respect to replication kinetics in vitro and pathogenicity and transmission in ferrets. Pandemic (H1N1) 2009 viruses containing basic polymerase 2 alone or in combination with acidic polymerase of seasonal (H1N1) virus were attenuated in ferrets. In contrast, pandemic (H1N1) 2009 with neuraminidase of seasonal (H3N2) virus resulted in increased virus replication and more severe pulmonary lesions. The data show that pandemic (H1N1) 2009 virus has the potential to reassort with seasonal influenza viruses, which may result in increased pathogenicity while it maintains the capacity of transmission through aerosols or respiratory droplets

Comparison of sequencing methods and data processing pipelines for whole genome sequencing and minority single nucleotide variant (mSNV) analysis during an influenza A/H5N8 outbreak

As high-throughput sequencing technologies are becoming more widely adopted for analysing pathogens in disease outbreaks there needs to be assurance that the different sequencing technologies and approaches to data analysis will yield reliable and comparable results. Conversely, understanding where agreement cannot be achieved provides insight into the limitations of these approaches and also allows efforts to be focused on areas of the process that need improvement. This manuscript describes the next-generation sequencing of three closely related viruses, each analysed using different sequencing strategies, sequencing instruments and data processing pipelines. In order to determine the comparability of consensus sequences and minority (sub-consensus) single nucleotide variant (mSNV) identification, the biological samples, the sequence data from 3 sequencing platforms and the *.bam quality-trimmed alignment files of raw data of 3 influenza A/H5N8 viruses were shared. This analysis demonstrated that variation in the final result could be attributed to all stages in the process, but the most critical were the well-known homopolymer errors introduced by 454 sequencing, and the alignment processes in the different data processing pipelines which affected the consistency of mSNV detection. However, homopolymer errors aside, there was generally a good agreement between consensus sequences that were obtained for all combinations of sequencing platforms and data processing pipelines. Nevertheless, minority variant analysis will need a different level of careful standardization and awareness about the possible limitations, as shown in this study

Lack of virological and serological evidence for continued circulation of highly pathogenic avian influenza H5N8 virus in wild birds in the Netherlands, 14 November 2014 to 31 January 2016

In 2014, H5N8 clade 2.3.4.4 highly pathogenic avian influenza (HPAI) viruses of the A/Goose/ Guangdong/1/1996 lineage emerged in poultry and wild birds in Asia, Europe and North America. Here, wild birds were extensively investigated in the Netherlands for HPAI H5N8 virus (real-time polymerase chain reaction targeting the matrix and H5 gene) and antibody detection (haemagglutination inhibition and virus neutralisation assays) before, during and after the first virus detection in Europe in late 2014. Between 21 February 2015 and 31 January 2016, 7,337 bird samples were tested for the virus. One HPAI H5N8 virus-infected Eurasian wigeon (Anas penelope) sampled on 25 February 2015 was detected. Serological assays were performed on 1,443 samples, including 149 collected between 2007 and 2013, 945 between 14 November 2014 and 13 May 2015, and 349 between 1 September and 31 December 2015. Antibodies specific for HPAI H5 clade 2.3.4.4 were absent in wild bird sera obtained before 2014 and present in sera collected during and after the HPAI H5N8 emergence in Europe, with antibody incidence declining after the 2014/15 winter. Our results indicate that the HPAI H5N8 virus has not continued to circulate extensively in wild bird populations since the 2014/15 winter and that independent maintenance of the virus in these populations appears unlikely

Detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction

We present two real-time reverse-transcription polymerase chain reaction assays for a novel human coronavirus (CoV), targeting regions upstream of the E gene (upE) or within open reading frame (ORF)1b, respectively. Sensitivity for upE is 3.4 copies per reaction (95% confidence interval (CI): 2.5-6.9 copies) or 291 copies/mL of sample. No cross-reactivity was observed with coronaviruses OC43, NL63, 229E, SARS-CoV, nor with 92 clinical specimens containing common human respiratory viruses. We recommend using upE for screening and ORF1b for confirmation

Human clade 2.3.4.4 A/H5N6 influenza virus lacks mammalian adaptation markers and does not transmit via the airborne route between ferrets

Since their emergence in 1997, A/H5N1 influenza viruses of the A/goose/ Guangdong/1/96 lineage have diversified in multiple genetic and antigenic clades upon continued circulation in poultry in several countries in Eurasia and Africa. Since 2009, reassortant viruses carrying clade 2.3.4.4 hemagglutinin (HA) and internal and neuraminidase (NA) genes of influenza A viruses of different avian origin have been detected, yielding various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8. Previous studies reported on the low pathogenicity and lack of airborne transmission of A/H5N2 and A/H5N8 viruses in the ferret model. However, although A/H5N6 viruses are the only clade 2.3.4.4 viruses that crossed the species barrier and infected humans, the risk they pose for human health remains poorly characterized. Here, the characterization of A/H5N6 A/Guangzhou/39715/2014 virus in vitro and in ferrets is described. This A/H5N6 virus possessed high polymerase activity, mediated by the E627K substitution in the PB2 protein, which corresponds to only one biological trait out of the three that were previously shown to confer airborne transmissibility to A/H5N1 viruses between ferrets. This might explain its lack of airborne transmission between ferrets. After intranasal inoculation, A/H5N6 virus replicated to high titers in the respiratory tracts of ferrets and was excreted for at least 6 days. Moreover, A/H5N6 virus caused severe pneumonia in ferrets upon intratracheal inoculation. Thus, A/H5N6 virus causes a more severe disease in ferrets than previously investigated clade 2.3.4.4 viruses, but our results demonstrate that the risk from airborne spread is currently low

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. Copyright