Genomic analysis of the population structure of Paenibacillus larvae in New Zealand.

New Zealand is a remote country in the South Pacific Ocean. The isolation and relatively late arrival of humans into New Zealand has meant there is a recorded history of the introduction of domestic species. Honey bees (Apis mellifera) were introduced to New Zealand in 1839, and the disease American foulbrood was subsequently found in the 1870s. Paenibacillus larvae, the causative agent of American foulbrood, has been genome sequenced in other countries. We sequenced the genomes of P. larvae obtained from 164 New Zealand apiaries where American foulbrood was identified in symptomatic hives during visual inspection. Multi-locus sequencing typing (MLST) revealed the dominant sequence type to be ST18, with this clonal cluster accounting for 90.2% of isolates. Only two other sequence types (with variants) were identified, ST5 and ST23. ST23 was only observed in the Otago area, whereas ST5 was limited to two geographically non-contiguous areas. The sequence types are all from the enterobacterial repetitive intergenic consensus I (ERIC I) genogroup. The ST18 and ST5 from New Zealand and international P. larvae all clustered by sequence type. Based on core genome MLST and SNP analysis, localized regional clusters were observed within New Zealand, but some closely related genomes were also geographically dispersed, presumably due to hive movements by beekeepers.

Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity.

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.

Substrate-dependent effects of quaternary structure on RNase E activity.

RNase E is an essential, multifunctional ribonuclease encoded in E. coli by the rne gene. Structural analysis indicates that the ribonucleolytic activity of this enzyme is conferred by rne-encoded polypeptide chains that (1) dimerize to form a catalytic site at the protein-protein interface, and (2) multimerize further to generate a tetrameric quaternary structure consisting of two dimerized Rne-peptide chains. We identify here a mutation in the Rne protein's catalytic region (E429G), as well as a bacterial cell wall peptidoglycan hydrolase (Amidase C [AmiC]), that selectively affect the specific activity of the RNase E enzyme on long RNA substrates, but not on short synthetic oligonucleotides, by enhancing enzyme multimerization. Unlike the increase in specific activity that accompanies concentration-induced multimerization, enhanced multimerization associated with either the E429G mutation or interaction of the Rne protein with AmiC is independent of the substrate's 5' terminus phosphorylation state. Our findings reveal a previously unsuspected substrate length-dependent regulatory role for RNase E quaternary structure and identify cis-acting and trans-acting factors that mediate such regulation.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

An alternative miRISC targets a cancer-associated coding sequence mutation in FOXL2.

Recent evidence suggests that animal microRNAs (miRNAs) can target coding sequences (CDSs); however, the pathophysiological importance of such targeting remains unknown. Here, we show that a somatic heterozygous missense mutation (c.402C>G; p.C134W) in FOXL2, a feature shared by virtually all adult-type granulosa cell tumors (AGCTs), introduces a target site for miR-1236, which causes haploinsufficiency of the tumor-suppressor FOXL2. This miR-1236-mediated selective degradation of the variant FOXL2 mRNA is preferentially conducted by a distinct miRNA-loaded RNA-induced silencing complex (miRISC) directed by the Argonaute3 (AGO3) and DHX9 proteins. In both patients and a mouse model of AGCT, abundance of the inversely regulated variant FOXL2 with miR-1236 levels is highly correlated with malignant features of AGCT. Our study provides a molecular basis for understanding the conserved FOXL2 CDS mutation-mediated etiology of AGCT, revealing the existence of a previously unidentified mechanism of miRNA-targeting disease-associated mutations in the CDS by forming a non-canonical miRISC.

Molecular Characterization and Identification of Calnexin 1 As a Radiation Biomarker from Tradescantia BNL4430.

Calnexin (CNX) is an integral membrane protein that functions as a chaperone in the endoplasmic reticulum for the correct folding of proteins under stress conditions, rendering organisms tolerant under adverse conditions. Studies have investigated the cytogenetic effects of gamma irradiation (Ɣ-IR) on plants, but information on the molecular response under Ɣ-IR remains limited. Previously, we constructed a cDNA library of an irradiation-sensitive bioindicator plant, Tradescantia BNL4430 (T-4430) under Ɣ-IR, in which the Calnexin-1 gene was highly upregulated at 50 mGy treatment. TrCNX1 encodes a 61.4 kDa protein with conserved signature motifs similar to already reported CNX1s. TrCNX1 expression was evaluated by semiquantitative reverse transcriptase PCR and quantitative real-time PCR and was ubiquitously expressed in various tissues and highly upregulated in flower petals under 50 mGy Ɣ-IR stress. The protective function of TrCNX1 was investigated by overexpression of TrCNX1 in an Escherichia coli BL21(DE3) heterologous system. Using plate assay, we showed that TrCNX1 increased the viability of E. coli transformants under both UV-B and Ɣ-IR compared with the control, demonstrating that TrCNX1 functions under irradiation stress. TrCNX1 may enhance irradiation stress tolerance in crops and act as a radio marker gene to monitor the effects of radiation.

Mechanisms of ß-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for ß-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for ß-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased ß-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased ß-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to ß-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis.

New Zealand rickettsia-like organism (NZ-RLO) and Tenacibaculum maritimum: Distribution and phylogeny in farmed Chinook salmon (Oncorhynchus tshawytscha).

A total of 777 fish from three growing regions of New Zealand Chinook salmon farms comprising of five sites were tested. Quantitative PCR was used to determine the distribution of New Zealand rickettsia-like organism and Tenacibaculum maritimum. Genetic information from these bacteria were then compared with strains reported worldwide. Using this information, suggested associations of pathogens with clinically affected fish were made. NZ-RLO was detected in two of the three regions, and T. maritimum was detected in all regions. Three strains of NZ-RLO were identified during this study. Based on analysis of the ITS rRNA gene, NZ-RLO1 appears to be part of an Australasian grouping sharing high similarity with the Tasmanian RLO, NZ-RLO2 was shown to be the same as an Irish strain, and NZ-RLO3 was shown be closely related to two strains from Chile. Based on multi-locus sequence typing, the New Zealand T. maritimum was the same as Australian strains. NZ-RLOs were detected more frequently in fish with skin ulcers than fish without skin ulcers. While additional research is required to investigate the pathogenicity of these organisms, this is the first time that NZ-RLOs have been associated with the development of clinical infections in farmed Chinook salmon.

Brucellosis in Endangered Hector's Dolphins (Cephalorhynchus hectori).

Brucella spp infections of marine mammals are often asymptomatic but have been associated with reproductive losses and deaths. Zoonotic infections originating from marine isolates have also been described. Hector's dolphins (Cephalorhynchus hectori) are an endangered species with a declining population, and the role of infectious disease in population dynamics is not fully understood. In this study, 27 Hector's dolphins found dead around the New Zealand coastline between November 2006 and October 2010 were evaluated for lesions previously associated with cetacean brucellosis. Tissues were examined using histological, immunohistochemical, and molecular (polymerase chain reaction [PCR]) techniques. Seven of 27 dolphins (26%) had at least 1 tissue that was positive on PCR for Brucella spp. Lesions consistent with brucellosis were present in 10 of 27 (37%) dolphins, but in 8 of these dolphins Brucella infection could not be demonstrated in lesional tissues. Two dolphins (7%) were diagnosed with active brucellosis: 1 female with placentitis and metritis, and 1 stillborn male fetus. Brucella identified in these 2 dolphins had genetic similarity (99%) to Brucella pinnipedialis. The omp2a gene amplicon from the uterus of the female had 100% homology with ST27 genotype isolates from a human in New Zealand and a bottlenose dolphin of Pacific origin. The remaining 5 PCR-positive dolphins were assessed as having asymptomatic or latent infection. While most Brucella infections identified in this study appeared to be subclinical, the finding of 2 dolphins with reproductive disease due to Brucella infection suggests that this disease has the potential to affect reproductive success in this species.

Investigation of a mortality cluster in wild adult yellow-eyed penguins (Megadyptes antipodes) at Otago Peninsula, New Zealand.

We investigated an epidemic mortality cluster of yellow-eyed penguins (Megadyptes antipodes) that involved 67 moribund or dead birds found on various beaches of the Otago Peninsula, New Zealand, between 21 January and 20 March 2013. Twenty-four carcases were examined post-mortem. Histological lesions of pulmonary, hepatic and splenic erythrophagocytosis and haemosiderosis were found in 23 of 24 birds. Fifteen birds also had haemoglobin-like protein droplets within renal tubular epithelial cells. Despite consistent histological lesions, a cause of death could not be established. Virology, bacteriology and molecular tests for avian influenza, avian paramyxovirus-1, avipoxvirus, Chlamydia psittaci, Plasmodium spp., Babesia spp., Leucocytozoon spp. and Toxoplasma gondii were negative. Tissue concentrations of a range of heavy metals (n = 4 birds) were consistent with low level exposure, while examination of proventricular contents and mucus failed to detect any marine biotoxins or Clostridium botulinum toxin. Hepatic concentrations of total polycyclic aromatic hydrocarbons (PAHs) (n = 5 birds) were similar to background concentrations of polycyclic aromatic hydrocarbons previously found in yellow-eyed penguins from the South Island of New Zealand, but there were significantly higher concentrations of 1-methylnapthelene and 2-methylnapthelene in the birds found dead in this mortality cluster. The biological significance of this finding is unclear. A temporal investigation of the epidemic did not indicate either a common source or propagative epidemic pattern. Although our investigation did not definitively implicate a toxic or infectious agent, we could not rule out causes such as toxic marine organisms or mycoplasmosis. Further investigations should therefore by carried out in the event of future mortality clusters.

Helicobacter bilis and Helicobacter trogontum: infectious causes of abortion in sheep.

The aim of our study was to determine the association of Helicobacter spp. that had flexispira morphology with ovine abortion, and to understand the importance of these organisms as a cause of ovine abortion in New Zealand. A retrospective diagnostic survey was carried out on laboratory submissions from ovine abortion outbreaks. A comparison was made of the proportion of laboratory submissions where Helicobacter spp. were detected from flocks that had no other agent identified (group A) with a group that had a known cause of abortion identified (group B). This latter group was considered to be a negative control, given the premise that Helicobacter spp. were not causing abortions and that Helicobacter spp. should be present at a lower rate in the group. Where no diagnosis had been made, aborted material was positive for Helicobacter spp. with flexispira morphology in 8 submissions (20%, 8/40) from 5 of the 31 survey farms (16%, 5/31). Helicobacter spp. were not detected in any of the 18 submissions from the 17 control farms (group B). Helicobacter spp. were confirmed by 16S ribosomal RNA sequencing of 3 of the Helicobacter spp. isolated by culture from the livers of aborted sheep fetuses, and 7 of the 8 where samples were positive in a Helicobacter PCR assay. The Helicobacter spp. were identified as Helicobacter trogontum (Flexispira taxon 5 genotype) and Helicobacter bilis (Flexispira taxon 8 genotype). The findings support Helicobacter spp. being a probable causative agent of ovine abortions in New Zealand.

Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast.

Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs.

Generation and analysis of expressed sequence tags (ESTs) of Camelina sativa to mine drought stress-responsive genes.

Camelina sativa is an oil-producing crop belonging to the family of Brassicaceae. Due to exceptionally high content of omega fatty acid, it is commercially grown around the world as edible oil, biofuel, and animal feed. A commonly referred 'false flax' or gold-of-pleasure Camelina sativa has been interested as one of biofuel feedstocks. The species can grow on marginal land due to its superior drought tolerance with low requirement of agricultural inputs. This crop has been unexploited due to very limited transcriptomic and genomic data. Use of gene-specific molecular markers is an important strategy for new cultivar development in breeding program. In this study, Illumina paired-end sequencing technology and bioinformatics tools were used to obtain expression profiling of genes responding to drought stress in Camelina sativa BN14. A total of more than 60,000 loci were assembled, corresponding to approximately 275 K transcripts. When the species was exposed to 10 kPa drought stress, 100 kPa drought stress, and rehydrated conditions, a total of 107, 2,989, and 982 genes, respectively, were up-regulated, while 146, 3,659, and 1189 genes, respectively, were down-regulated compared to control condition. Some unknown genes were found to be highly expressed under drought conditions, together with some already reported gene families such as senescence-associated genes, CAP160, and LEA under 100 kPa soil water condition, cysteine protease, 2OG, Fe(II)-dependent oxygenase, and RAD-like 1 under rehydrated condition. These genes will be further validated and mapped to determine their function and loci. This EST library will be favorably applied to develop gene-specific molecular markers and discover genes responsible for drought tolerance in Camelina species.

Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach.

Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at http://pombe.kaist.ac.kr/compendium.

Evaluation of the pathogenicity of avipoxvirus strains isolated from wild birds in New Zealand and the efficacy of a fowlpox vaccine in passerines.

Avipoxvirus (APV) infection is a highly contagious disease of birds and has been reported in more than 200 bird species, affecting both domesticated and free-ranging birds around the world. In New Zealand, at least three different strains of Avipoxvirus (APV) have been identified in a range of bird species.The pathogenicityof two APV strains isolated from wild birds in New Zealand, representing subclade A1 and subclade B1 were compared in zebra finches (Taeniopygia guttata). The efficacy of fowlpox vaccine at preventing clinical disease in passerines was also evaluated. Twenty-five zebra finches were divided into five groups (I-IV and a control group). Birds from Groups II and IV were vaccinated using fowl poxvirus vaccine prior to challenge. Subsequently two groups (I and II) were inoculated with a silvereye isolate (A1) and the other two groups (Group III and IV) were inoculated with a blackbird isolate (B1). Both inocula were previously propagated in chicken fibroblast cell culture. Birds in the control group were inoculated with sterile PBS. Skin thickness at the inoculation sites was measured and the development of additional skin lesions was monitored. Antibody development was measured by ELISA pre- and post virus inoculation. Both APV strains caused either swelling or hyperplasia at the inoculation site of non-vaccinated birds (4/5 in Group I and 5/5 in Group III). The swelling was milder and no foot lesions were observed in vaccinated birds before or after challenge with the silvereye or blackbird APV strains. These findings indicated that the fowlpox vaccine provided safe and appropriate protection for zebra finches exposed to the two wild APV strains and suggest that the vaccine has the potential to be used where APV threatens the captive management or translocation of endangered passerines.

Identification of a hyperactive variant of the SecM motif involved in ribosomal arrest.

Recent studies in several organisms have shown that certain nascent sticky peptides stall in the ribosome during their own translation. Amino acid sequences present at the C-terminal part of Escherichia coli SecM ((150)FSTPVWISQAQGIRAGP(166)) have a well-characterized role in ribosome stalling. To investigate the determinants of the SecM motif responsible for ribosome stalling, we performed a genetic screen for mutants with an altered SecM motif that resulted in altered ribosome stalling. To do this, we used a cat fusion construct containing the SecM motif and a myc-tag (cat'-'myc-secM). This construct expresses cat'-'myc-secM mRNA transcripts predominantly translated by a subset of ribosomes called specialized ribosomes that recognize an altered ribosome binding sequence in the mRNA. While all of the isolated mutants containing mutations at the functionally conserved amino acid residues at positions between 161 and 166 showed decreased ribosome stalling, one mutant sequence containing an amino acid substitution from serine to lysine at position 157 (S157K) showed enhanced ribosome stalling that consequently increased mRNA cleavage. Our results reveal that a functionally not conserved amino acid residue at position 157 of SecM can also affect ribosome stalling and provide additional insight into the molecular mechanisms underlying sticky-peptide-induced ribosome arrest.

The phylogenetic analysis of avipoxvirus in New Zealand.

Avipoxvirus is known to be endemic in New Zealand and it is a cause of ongoing mortalities in the endangered black robin and shore plover populations. There is no information on the strains of avipoxvirus occurring in New Zealand and their likely origin or pathogenicity. This study was designed to identify the phylogenetic relationships of pathogenic avipoxvirus strains infecting introduced, native, and endemic bird species in New Zealand. Avipoxvirus 4b core protein gene was detected in tissue samples from 25/48 birds (52.1%) from 15 different species in New Zealand. Bootstrap analysis of avipoxvirus 4b core protein gene revealed that the New Zealand avipoxvirus isolates comprised of three different subclades. The majority of New Zealand avipoxvirus isolates (74%) belonged to A1 subclade which shared 100% genetic similarity with the fowlpox HPB strain. An isolate from a wood-pigeon (kereru) belonged to subclade A3, displaying 100% sequence homology to albatrosspox virus. An additional group, isolated from two shore plovers and one South Island saddleback, grouped within subclade B1 and presented 99% sequence homology to European PM33/2007 and Hawaiian HAAM 22.10H8 isolates. The results suggest that a variety of New Zealand bird species are susceptible to avipoxvirus infection, that there are more than two distinctive avipoxvirus subclades in New Zealand, and that the most prevalent A1 strain may have been introduced to New Zealand through introduced avian hosts such as passerines or poultry.

Functional study of the residue C899 in the 900 tetraloop of Escherichia coli small subunit ribosomal RNA.

A mutant ribosome bearing C899G in the 900 tetraloop of Escherichia coli 16S rRNA, one implicated in a conformational switch in the dynamic movements of the ribosome, showed defects in subunit association and 30S initiation complex formation. Our results explain the basis of the loss of protein synthesis ability caused by a perturbation of the 900 tetraloop.