Matthew Peacey

Evaluation of rapid and simple techniques for the enrichment of viruses prior to metagenomic virus discovery

Abstract The discovery of new or divergent viruses using metagenomics and high-throughput sequencing has become more commonplace. The preparation of a sample is known to have an effect on the representation of virus sequences within the metagenomic dataset yet comparatively little attention has been given to this. Physical enrichment techniques are often applied to samples to increase the number of viral sequences and therefore enhance the probability of detection. With the exception of virus ecology studies, there is a paucity of information available to researchers on the type of sample preparation required for a viral metagenomic study that seeks to identify an aetiological virus in an animal or human diagnostic sample. A review of published virus discovery studies revealed the most commonly used enrichment methods, that were usually quick and simple to implement, namely low-speed centrifugation, filtration, nuclease-treatment (or combinations of these) which have been routinely used but often without justification. These were applied to a simple and well-characterised artificial sample composed of bacterial and human cells, as well as DNA (adenovirus) and RNA viruses (influenza A and human enterovirus), being either non-enveloped capsid or enveloped viruses. The effect of the enrichment method was assessed by both quantitative real-time PCR and metagenomic analysis that incorporated an amplification step. Reductions in the absolute quantities of bacteria and human cells were observed for each method as determined by qPCR, but the relative abundance of viral sequences in the metagenomic dataset remained largely unchanged. A 3-step method of centrifugation, filtration and nuclease-treatment showed the greatest increase in the proportion of viral sequences. This study provides a starting point for the selection of a purification method in future virus discovery studies, and highlights the need for more data to validate the effect of enrichment methods on different sample types, amplification, bioinformatics approaches and sequencing platforms. This study also highlights the potential risks that may attend selection of a virus enrichment method without any consideration for the sample type being investigated.

Pandemic (H1N1) 2009 and seasonal influenza a (H1N1) co-infection, New Zealand, 2009

Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility.Co-infection with seasonal influenza A (H1N1) and pandemic (H1N1) 2009 could result in reassortant viruses that may acquire new characteristics of transmission, virulence, and oseltamivir susceptibility. Results from oseltamivir-sensitivity testing on viral culture suggested the possibility of co-infections with oseltamivir-resistant (seasonal A [H1N1]) and -susceptible (pandemic [H1N1] 2009) viruses.

Virus-like particles from rabbit hemorrhagic disease virus can induce an anti-tumor response

Recombinant virus-like particles (VLP) expressing heterologous tumor antigens have recently been investigated for use as vaccines. We have chemically conjugated ovalbumin (OVA) or OVA-derived CD4 (OTII) and CD8 (OTI) epitopes, to rabbit hemorrhagic disease virus (RHDV) VLP. VLP conjugated with OVA were able to cross-prime CD8+ cells from OT1 mice transgenic for the OVA T cell receptor. VLP.OTI was able to induce higher antigen-specific cytotoxicity in vivo than VLP mixed with either the protein or the peptide. Furthermore we have shown that the growth of the aggressive B16.OVA melanoma in mice was significantly delayed in those animals that had been vaccinated with VLP.OVA or with VLP coupled with both OTI and OTII peptides prior to the introduction of the tumor. Neither VLP.OTI nor VLP.OTII alone were capable of inhibiting tumor growth. This work suggests that RHDV VLP offer a versatile scaffold for multiple vaccine epitopes, enabling cross-presentation of the antigen to elicit potent cell-mediated and anti-tumor responses.

Metagenomic Detection of Viruses in Aerosol Samples from Workers in Animal Slaughterhouses

Published studies have shown that workers in animal slaughterhouses are at a higher risk of lung cancers as compared to the general population. No specific causal agents have been identified, and exposures to several chemicals have been examined and found to be unrelated. Evidence suggests a biological aetiology as the risk is highest for workers who are exposed to live animals or to biological material containing animal faeces, urine or blood. To investigate possible biological exposures in animal slaughterhouses, we used a metagenomic approach to characterise the profile of organisms present within an aerosol sample. An assessment of aerosol exposures for individual workers was achieved by the collection of personal samples that represent the inhalable fraction of dust/bioaerosol in workplace air in both cattle and sheep slaughterhouses. Two sets of nine personal aerosol samples were pooled for the cattle processing and sheep processing areas respectively, with a total of 332,677,346 sequence reads and 250,144,492 sequence reads of 85 bp in length produced for each. Eukaryotic genome sequence was found in both sampling locations, and bovine, ovine and human sequences were common. Sequences from WU polyomavirus and human papillomavirus 120 were detected in the metagenomic dataset from the cattle processing area, and these sequences were confirmed as being present in the original personal aerosol samples. This study presents the first metagenomic description of personal aerosol exposure and this methodology could be applied to a variety of environments. Also, the detection of two candidate viruses warrants further investigation in the setting of occupational exposures in animal slaughterhouses.

Genotyping assay for the identification of 2009–2010 pandemic and seasonal H1N1 influenza virus reassortants

New Zealand identified its first pandemic H1N1 influenza cases in late April 2009, immediately prior to the historical start of the New Zealand influenza season. Both pandemic and oseltamivir-resistant seasonal H1N1 viruses cocirculated in the population for a period of time. Thus, concerns were raised about the possibility of reassortment events between the two strains. An RT-PCR-based genotyping assay was developed so that H1N1 influenza coinfections and reassortants could be detected quickly. The assay differentiated effectively the seasonal and pandemic strains. It also confirmed the identification of the first reported coinfection of pandemic and seasonal H1N1 strains during the 2009 Southern Hemisphere influenza season in New Zealand.

New Alphacoronavirus in Mystacina tuberculata Bats, New Zealand

Because of recent interest in bats as reservoirs of emerging diseases, we investigated the presence of viruses in Mystacina tuberculata bats in New Zealand. A novel alphacoronavirus sequence was detected in guano from roosts of M. tuberculata bats in pristine indigenous forest on a remote offshore island (Codfish Island).

Diagnostic assay recommended by the world health organization for swine origin influenza a (h1n1) virus cross-reacts with H5N1 influenza virus.

Pandemic influenza poses a significant risk to global human health. The unpredictable nature of the influenza virus necessitates accurate, reliable, and rapid diagnostic assays for the detection of, and characterization of the current pandemic caused by, swine origin influenza virus (SOIV) in order to better understand the nature of the pandemic and implement appropriate health interventions. SOIV emerged in mid-April 2009 (1), leaving laboratories around the world scrambling to establish a diagnostic test to detect this novel influenza virus. The World Health Organization (WHO) responded with remarkable speed by releasing guidelines and protocols for a real-time reverse transcription (RT)-PCR assay 15 days after the reported identification of SOIV (2). This provided a rapid and sensitive assay for the detection of SOIV that was critical for the determination of the spread and extent of SOIV infections. These guidelines recommend three primer-and-probe sets: InfA, amplifying a conserved region of the matrix gene from all influenza A viruses; SW H1, designed to specifically detect the hemagglutinin gene segment (subtype H1) from SOIV; and SW InfA, designed to specifically detect the nucleoprotein (NP) gene segment from all swine influenza viruses. As part of the WHO Global Influenza Surveillance Network, the New Zealand National Influenza Centre regularly participates in WHO external quality assurance panels of 10 RNA samples that are H1N1, H3N2, or H5N1 influenza A virus or, recently, SOIV. In following the WHO recommended guidelines exactly, it was found that the SW InfA primer-and-probe set amplified all six H5N1 RNA samples. This is clinically relevant, as the proficiency panel RNA returned threshold cycle (CT) values (InfA assay, 23.44 to 28.67) well within the range of what we regularly observe with clinical samples. In addition, the SW InfA primer-and-probe set detected two additional H5N1 viruses not provided in the proficiency panel, the human H5N1 strain A/Vietnam/3028/2004 and an avian H5N1 strain, A/Mallard/New Zealand/272-73/2008. The efficiency of this reaction was 79% (three replicates) compared to 94% (five replicates) for the amplification of SOIV RNA, suggesting that mismatches within the primer target region reduced assay efficiency. To prove that the H5N1 NP gene sequence was amplified by the SOIV SW InfA assay, all real-time RT-PCR amplicons were sequenced. The two SOIV RNA samples from the proficiency panel and SOIV-positive control showed 100% identity to the consensus sequence of 394 NP full-length gene segments available as of 29 July 2009. In contrast, all H5N1 RNA samples from the proficiency panel contained at least 31 nucleotide differences from the SOIV consensus sequence but showed between 93.3% and 96.8% identity to A/Vietnam/1203/2004. Alignment of all SOIV and human H5N1 NP gene segments available in GenBank showed that 391 SOIV viruses have one mismatch in the SW InfA forward primer (Table ​(Table1).1). The remaining three SOIV sequences have a further mismatch in either the forward primer or the probe. The majority (74.6%, or 91 sequences) of available human H5N1 sequences are identical within the SW InfA primer-and-probe regions, having two mismatches in the forward primer, two in the probe, and four in the reverse primer region. None of the mismatches occurred within 9 nucleotides of the 3′ end, an important determinant for primer specificity. TABLE 1. Comparison of SOIV and human H5N1 NP gene sequences with SW infA primer and probe sequences The speed at which these assays were developed and deployed to the WHO Global Influenza Surveillance Network was remarkable, particularly with the limited SOIV sequence information available at the time of development. Other factors which constrained the development of RT-PCR primer targets include the origin and genetic rate of change for each gene segment; only five of the eight gene segments are of swine origin (3), while the rapidly evolving HA and NA genes have a propensity to drift at a much higher rate than conserved segments such as NP (5). This report demonstrates that the SW InfA assay is not specific to SOIV and is able to detect both human and avian (H5N1) influenza A viruses and so there is the potential for misidentification. Following WHO guidelines, the utilization of all primer-and-probe sets for the detection of SOIV should provide an accurate diagnosis. However, we have observed that for the same SOIV RNA sample, the SW InfA assay consistently returns lower CT values than the SW H1 assay and so clinical samples with higher CT values can yield an SW H1-negative, SW InfA-positive result. In these cases, we suggest sequencing of real-time SW InfA PCR amplicons to ensure the amplification of SOIV (4). Alternatively, and especially for countries in which H5N1 is endemic, an H5N1-specific assay could be performed to exclude this diagnosis. However, a negative H5N1 result would not exclude other influenza virus subtypes that may cross-react with the SOIV assay.

Immunogenicity and protective efficacy of mycobacterial DNA vaccines incorporating plasmid-encoded cytokines against Mycobacterium bovis.

DNA‐based vaccines, alone or in combination with other sub‐unit vaccination regimes, represent an alternative to live mycobacterial vaccines for protective immunization against tuberculosis. Here, we have used a murine immunization or Mycobacteriam bovis aerosol challenge model to assess the immunogenicity and protective efficacy of mycobacterial DNA vaccines. Mice that received immunization with DNA constructs encoding M. bovis antigen 85A (Ag85–A) and arget(ESAT‐6) produced measurable interferon‐gamma (IFN‐γ) responses to CD4+ T‐cell epitope‐peptide recall antigens in vitro. The magnitude of these responses was enhanced by co‐delivery of a construct encoding murine cytokines (macrophage inhibitory protein (MIP)‐1α or interleukin(IL)‐7), although they did not the match responses observed in mice that received Bacille Calmette−Guerin(BCG) immunisation. In contrast, DNA priming followed by boosting with modified vaccinia Ankara (MVA) vaccine (expressing M. tuberculosis Ag85–A) invoked higher IFN‐γ levels, with the most immunogenic regime of Ag85 or ESAT or IL‐7 prime followed by MVA boost being of commensurate immunogenicity to BCG. Despite this, neither DNA alone nor DNA‐prime or MVA boost regimes conferred measurable protection against aerosol challenge with virulent M. bovis. These data highlight both the promise and the shortcomings of new generation subunit tuberculosis vaccines, with particular emphasis on their potential as vaccines against M. bovis.

Enterovirus 74 Infection in Children

Enterovirus 74 (EV74) is a rarely detected viral infection of children. In 2010, EV74 was identified in New Zealand in a 2 year old child with acute flaccid paralysis (AFP) through routine polio AFP surveillance. A further three cases of EV74 were identified in children within six months. These cases are the first report of EV74 in New Zealand. In this study we describe the near complete genome sequence of four EV74 isolates from New Zealand, which shows only limited sequence identity in the non-structural proteins when compared to the other two known EV74 sequences. As is typical of enteroviruses multiple recombination events were evident, particularly in the P2 region and P3 regions. This is the first complete EV74 genome sequenced from a patient with acute flaccid paralysis.

Pandemic Seasonal H1N1 Reassortants Recovered from Patient Material Display a Phenotype Similar to That of the Seasonal Parent

ABSTRACT We have previously shown that 11 patients became naturally coinfected with seasonal H1N1 (A/H1N1) and pandemic H1N1 (pdm/H1N1) during the Southern hemisphere winter of 2009 in New Zealand. Reassortment of influenza A viruses is readily observed during coinfection of host animals and in vitro; however, reports of reassortment occurring naturally in humans are rare. Using clinical specimen material, we show reassortment between the two coinfecting viruses occurred with high likelihood directly in one of the previously identified patients. Despite the lack of spread of these reassortants in the community, we did not find them to be attenuated in several model systems for viral replication and virus transmission: multistep growth curves in differentiated human bronchial epithelial cells revealed no growth deficiency in six recovered reassortants compared to A/H1N1 and pdm/H1N1 isolates. Two reassortant viruses were assessed in ferrets and showed transmission to aerosol contacts. This study demonstrates that influenza virus reassortants can arise in naturally coinfected patients. IMPORTANCE Reassortment of influenza A viruses is an important driver of virus evolution, but little has been done to address humans as hosts for the generation of novel influenza viruses. We show here that multiple reassortant viruses were generated during natural coinfection of a patient with pandemic H1N1 (2009) and seasonal H1N1 influenza A viruses. Though apparently fit in model systems, these reassortants did not become established in the wider population, presumably due to herd immunity against their seasonal H1 antigen.