Mosquito-Borne Viruses and Insect-Specific Viruses Revealed in Field-Collected Mosquitoes by a Monitoring Tool Adapted from a Microbial Detection Array.

Several mosquito-borne diseases affecting humans are emerging or reemerging in the United States. The early detection of pathogens in mosquito populations is essential to prevent and control the spread of these diseases. In this study, we tested the potential applicability of the Lawrence Livermore Microbial Detection Array (LLMDA) to enhance biosurveillance by detecting microbes present in Aedes aegypti, Aedes albopictus, and Culex mosquitoes, which are major vector species globally, including in Texas. The sensitivity and reproducibility of the LLMDA were tested in mosquito samples spiked with different concentrations of dengue virus (DENV), revealing a detection limit of >100 but <1,000 PFU/ml. Additionally, field-collected mosquitoes from Chicago, IL, and College Station, TX, of known infection status (West Nile virus [WNV] and Culex flavivirus [CxFLAV] positive) were tested on the LLMDA to confirm its efficiency. Mosquito field samples of unknown infection status, collected in San Antonio, TX, and the Lower Rio Grande Valley (LRGV), TX, were run on the LLMDA and further confirmed by PCR or quantitative PCR (qPCR). The analysis of the field samples with the LLMDA revealed the presence of cell-fusing agent virus (CFAV) in A. aegypti populations. Wolbachia was also detected in several of the field samples (A. albopictus and Culex spp.) by the LLMDA. Our findings demonstrated that the LLMDA can be used to detect multiple arboviruses of public health importance, including viruses that belong to the Flavivirus, Alphavirus, and Orthobunyavirus genera. Additionally, insect-specific viruses and bacteria were also detected in field-collected mosquitoes. Another strength of this array is its ability to detect multiple viruses in the same mosquito pool, allowing for the detection of cocirculating pathogens in an area and the identification of potential ecological associations between different viruses. This array can aid in the biosurveillance of mosquito-borne viruses circulating in specific geographical areas.IMPORTANCE Viruses associated with mosquitoes have made a large impact on public and veterinary health. In the United States, several viruses, including WNV, DENV, and chikungunya virus (CHIKV), are responsible for human disease. From 2015 to 2018, imported Zika cases were reported in the United States, and in 2016 to 2017, local Zika transmission occurred in the states of Texas and Florida. With globalization and a changing climate, the frequency of outbreaks linked to arboviruses will increase, revealing a need to better detect viruses in vector populations. With the capacity of the LLMDA to detect viruses, bacteria, and fungi, this study highlights its ability to broadly screen field-collected mosquitoes and contribute to the surveillance and management of arboviral diseases.

Molecular evidence of viral DNA in non-small cell lung cancer and non-neoplastic lung.

BACKGROUND: Although ∼20% of human cancers are caused by microorganisms, only suspicion exists for a microbial cause of lung cancer. Potential infectious agents were investigated in non-small cell lung cancer (NSCLC) and non-neoplastic lung. METHODS: Seventy NSCLC tumours (33 squamous cell carcinomas, 17 adenocarcinomas, 10 adenocarcinomas with lepidic spread, and 10 oligometastases) and 10 non-neoplastic lung specimens were evaluated for molecular evidence of microorganisms. Tissues were subjected to the Lawrence Livermore Microbial Detection Array, an oncovirus panel of the International Agency for Research on Cancer, and human papillomavirus (HPV) genotyping. Associations were examined between microbial prevalence, clinical characteristics, and p16 and EGFR expression. RESULTS: Retroviral DNA was observed in 85% squamous cell carcinomas, 47% adenocarcinomas, and 10% adenocarcinomas with lepidic spread. Human papillomavirus DNA was found in 69% of squamous cell carcinomas with 30% containing high-risk HPV types. No significant viral DNA was detected in non-neoplastic lung. Patients with tumours containing viral DNA experienced improved long-term survival compared with patients with viral DNA-negative tumours. CONCLUSIONS: Most squamous cell carcinomas and adenocarcinomas contained retroviral DNA and one-third of squamous cell carcinomas contained high-risk HPV DNA. Viral DNA was absent in non-neoplastic lung. Trial results encourage further study of the viral contribution to lung carcinogenesis.

Metagenomic analysis of the airborne environment in urban spaces.

The organisms in aerosol microenvironments, especially densely populated urban areas, are relevant to maintenance of public health and detection of potential epidemic or biothreat agents. To examine aerosolized microorganisms in this environment, we performed sequencing on the material from an urban aerosol surveillance program. Whole metagenome sequencing was applied to DNA extracted from air filters obtained during periods from each of the four seasons. The composition of bacteria, plants, fungi, invertebrates, and viruses demonstrated distinct temporal shifts. Bacillus thuringiensis serovar kurstaki was detected in samples known to be exposed to aerosolized spores, illustrating the potential utility of this approach for identification of intentionally introduced microbial agents. Together, these data demonstrate the temporally dependent metagenomic complexity of urban aerosols and the potential of genomic analytical techniques for biosurveillance and monitoring of threats to public health.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing.

Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Association of Kaposi's sarcoma-associated herpesvirus (KSHV) with bladder cancer in Croatian patients.

As the seventh most common human malignancy, bladder cancer represents a global health problem. In addition to well-recognized risk factors such as smoking and exposure to chemicals, various infectious agents have been implicated as cofactors in the pathogenesis of urothelial malignancies. The aim of the present study was to assess the possible association of viral infection and bladder cancer in Croatian patients. Biopsy specimens were collected from a total of 55 patients diagnosed with different stages of bladder cancer. Initial screening of DNA extracts for the presence of viruses on Lawrence Livermore Microbial Detection Array revealed Kaposi's sarcoma-associated herpesvirus (KSHV) in each of three randomly chosen biopsy specimens. The prevalence of infection with KSHV among study population was then examined by KSHV-specific polymerase chain reaction (PCR) and immunoblotting. By nested PCR, KSHV DNA was detected in 55% of patients. KSHV, also known as human herpesvirus 8, is an infectious agent known to cause cancer. Its oncogenic potential is primarily recognized from its role in Kaposi's sarcoma, but it has also been involved in pathogenesis of two lymphoproliferative disorders. A high prevalence of KSHV infection in our study indicates that KSHV may play a role in tumorigenesis of bladder cancer and warrants further studies.

Metagenomic features of bioburden serve as outcome indicators in combat extremity wounds.

Battlefield injury management requires specialized care, and wound infection is a frequent complication. Challenges related to characterizing relevant pathogens further complicates treatment. Applying metagenomics to wounds offers a comprehensive path toward assessing microbial genomic fingerprints and could indicate prognostic variables for future decision support tools. Wound specimens from combat-injured U.S. service members, obtained during surgical debridements before delayed wound closure, were subjected to whole metagenome analysis and targeted enrichment of antimicrobial resistance genes. Results did not indicate a singular, common microbial metagenomic profile for wound failure, instead reflecting a complex microenvironment with varying bioburden diversity across outcomes. Genus-level Pseudomonas detection was associated with wound failure at all surgeries. A logistic regression model was fit to the presence and absence of antimicrobial resistance classes to assess associations with nosocomial pathogens. A. baumannii detection was associated with detection of genomic signatures for resistance to trimethoprim, aminoglycosides, bacitracin, and polymyxin. Machine learning classifiers were applied to identify wound and microbial variables associated with outcome. Feature importance rankings averaged across models indicated the variables with the largest effects on predicting wound outcome, including an increase in P. putida sequence reads. These results describe the microbial genomic determinants in combat wound bioburden and demonstrate metagenomic investigation as a comprehensive tool for providing information toward aiding treatment of combat-related injuries.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

A microbial detection array (MDA) for viral and bacterial detection.

BACKGROUND: Identifying the bacteria and viruses present in a complex sample is useful in disease diagnostics, product safety, environmental characterization, and research. Array-based methods have proven utility to detect in a single assay at a reasonable cost any microbe from the thousands that have been sequenced. METHODS: We designed a pan-Microbial Detection Array (MDA) to detect all known viruses (including phages), bacteria and plasmids and developed a novel statistical analysis method to identify mixtures of organisms from complex samples hybridized to the array. The array has broader coverage of bacterial and viral targets and is based on more recent sequence data and more probes per target than other microbial detection/discovery arrays in the literature. Family-specific probes were selected for all sequenced viral and bacterial complete genomes, segments, and plasmids. Probes were designed to tolerate some sequence variation to enable detection of divergent species with homology to sequenced organisms, and to have no significant matches to the human genome sequence. RESULTS: In blinded testing on spiked samples with single or multiple viruses, the MDA was able to correctly identify species or strains. In clinical fecal, serum, and respiratory samples, the MDA was able to detect and characterize multiple viruses, phage, and bacteria in a sample to the family and species level, as confirmed by PCR. CONCLUSIONS: The MDA can be used to identify the suite of viruses and bacteria present in complex samples.

Manipulation of the Gut Microbiome Alters Acetaminophen Biodisposition in Mice.

The gut microbiota is a vast and diverse microbial community that has co-evolved with its host to perform a variety of essential functions involved in the utilization of nutrients and the processing of xenobiotics. Shifts in the composition of gut microbiota can disturb the balance of organisms which can influence the biodisposition of orally administered drugs. To determine how changes in the gut microbiome can alter drug disposition, the pharmacokinetics (PK), and biodistribution of acetaminophen were assessed in C57Bl/6 mice after treatment with the antibiotics ciprofloxacin, amoxicillin, or a cocktail of ampicillin/neomycin. Altered PK, and excretion profiles of acetaminophen were observed in antibiotic exposed animals. Plasma Cmax was significantly decreased in antibiotic treated animals suggesting decreased bioavailability. Urinary metabolite profiles revealed decreases in acetaminophen-sulfate metabolite levels in both the amoxicillin and ampicillin/neomycin treated animals. The ratio between urinary and fecal excretion was also altered in antibiotic treated animals. Analysis of gut microbe composition revealed that changes in microbe content in antibiotic treated animals was associated with changes in acetaminophen biodisposition. These results suggest that exposure to amoxicillin or ampicillin/neomycin can alter the biodisposition of acetaminophen and that these alterations could be due to changes in gut microbiome composition.

Axiom Microbiome Array, the next generation microarray for high-throughput pathogen and microbiome analysis.

Microarrays have proven to be useful in rapid detection of many viruses and bacteria. Pathogen detection microarrays have been used to diagnose viral and bacterial infections in clinical samples and to evaluate the safety of biological drug materials. In this study, the Axiom Microbiome Array was evaluated to determine its sensitivity, specificity and utility in microbiome analysis of veterinary clinical samples. The array contains probes designed to detect more than 12,000 species of viruses, bacteria, fungi, protozoa and archaea, yielding the most comprehensive microbial detection platform built to date. The array was able to detect Shigella and Aspergillus at 100 genome copies, and vaccinia virus DNA at 1,000 genome copies. The Axiom Microbiome Array made correct species-level calls in mock microbial community samples. When tested against serum, tissue, and fecal samples from pigs experimentally co-infected with porcine reproductive and respiratory syndrome virus and porcine circovirus type 2, the microarray correctly detected these two viruses and other common viral and bacterial microbiome species. This cost-effective and high-throughput microarray is an efficient tool to rapidly analyze large numbers of clinical and environmental samples for the presence of multiple viral and bacterial pathogens.

Nonstructural proteins nsp2TF and nsp2N of porcine reproductive and respiratory syndrome virus (PRRSV) play important roles in suppressing host innate immune responses.

Recently, we identified a unique -2/-1 ribosomal frameshift mechanism in PRRSV, which yields two truncated forms of nonstructural protein (nsp) 2 variants, nsp2TF and nsp2N. Here, in vitro expression of individual PRRSV nsp2TF and nsp2N demonstrated their ability to suppress cellular innate immune responses in transfected cells. Two recombinant viruses were further analyzed, in which either nsp2TF was C-terminally truncated (vKO1) or expression of both nsp2TF and nsp2N was knocked out (vKO2). Host cellular mRNA profiling showed that a panel of cellular immune genes, in particular those involved in innate immunity, was upregulated in cells infected with vKO1 and vKO2. Compared to the wild-type virus, vKO1 and vKO2 expedited the IFN-α response and increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Our data strongly implicate nsp2TF/nsp2N in arteriviral immune evasion and demonstrate that nsp2TF/nsp2N-deficient PRRSV is less capable of counteracting host innate immune responses.

Double-stranded viral RNA persists in vitro and in vivo during prolonged infection of porcine reproductive and respiratory syndrome virus.

In order to study the mechanism of PRRSV persistence, an in vitro model of persistence was developed by serially passaging PRRSV-infected MARC-145 cells 109 times. Viral persistence was detected to be associated with increased double-stranded (dsRNA) in the infected cells. In PRRSV infected pigs, reduced ratio of plus to minus strands of viral RNA was observed in lymphoid tissues from PRRSV persistent pigs at 52 days post infection. Viral dsRNA was mostly detected in the germinal center during persistent infection compared to the localization of dsRNA in the inter-follicular zones during acute infection. RNA array analysis of antiviral cytokines in persistently infected lymph nodes showed that the presence of dsRNA did not stimulate antiviral immunity. These results suggest that PRRSV dsRNA functions as a mediator for viral persistence. The localization of PRRSV dsRNA in the germinal center of lymphoid tissues reveals a novel mechanism for PRRSV persistence.

Increased microbiome diversity at the time of infection is associated with improved growth rates of pigs after co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2).

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most important pathogens affecting the swine industry worldwide. Co-infections are common on a global scale, resulting in pork production losses through reducing weight gain and causing respiratory disease in growing pigs. Our initial work demonstrated that the fecal microbiome was associated with clinical outcome of pigs 70days post-infection (dpi) with PRRSV and PCV2. However, it remained uncertain if microbiome characteristics could predispose response to viral infection. The purpose of this study was to determine if microbiome characteristics present at the time of virus exposure were associated with outcome after co-infection. Using the Lawrence Livermore Microbial Detection Array, we profiled the microbiome in feces prior to infection from pigs identified retrospectively as having high or low growth rates after co-infection. High growth rate pigs had less severe interstitial pneumonia, reduced virus replication, and a significant increase in average daily weight gain throughout the study. At the level of the fecal microbiome, high growth rate pigs had increased microbial diversity on both a family and species level. Shifts in the microbiome composition of high growth rate pigs included reduced Methanobacteriaceae species, increased Ruminococcaceae species, and increased Streptococcaceae species when compared to low growth rate pigs. The results indicate that both microbiome diversity and composition at the time of virus exposure may play a role in the subsequent response of pigs to PRRSV/PCV2 co-infection.

Microbiome associations in pigs with the best and worst clinical outcomes following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2).

On a world-wide basis, co-infections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are common and contribute to a range of polymicrobial disease syndromes in swine. Both viruses compromise host defenses, resulting in increased susceptibility to infections by primary and secondary pathogens that can affect growth performance as well as increased morbidity and mortality. An experimental population of 95 pigs was co-infected with PRRSV and PCV2. At 70days post-infection (dpi), 20 representative pigs were selected as having the best or worst clinical outcome based on average daily gain (ADG) and the presence of clinical disease. Worst clinical outcome pigs had prolonged and greater levels of viremia as measured by qPCR. Serum, lung and fecal samples collected at 70 dpi were analyzed using a comprehensive DNA microarray technology, the Lawrence Livermore Microbial Detection Array, to detect over 8000 microbes. Bacterial species, such as Bacillus cereus, were detected at a higher rate in the serum of worst performing pigs. At the level of the fecal microbiome, the overall microbial diversity was lower in the worst clinical outcome group. The results reinforce the importance of pathogen load in determining clinical outcome and suggest an important role of microbial diversity as a contributing factor in disease.

Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing.

Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms.

Application of a pathogen microarray for the analysis of viruses and bacteria in clinical diagnostic samples from pigs.

Many of the disease syndromes challenging the commercial swine industry involve the analysis of complex problems caused by polymicrobial, emerging or reemerging, and transboundary pathogens. This study investigated the utility of the Lawrence Livermore Microbial Detection Array (Lawrence Livermore National Laboratory, Livermore, California), designed to detect 8,101 species of microbes, in the evaluation of known and unknown microbes in serum, oral fluid, and tonsil from pigs experimentally coinfected with Porcine reproductive and respiratory syndrome virus (PRRSV) and Porcine circovirus-2 (PCV-2). The array easily identified PRRSV and PCV-2, but at decreased sensitivities compared to standard polymerase chain reaction detection methods. The oral fluid sample was the most informative, possessing additional signatures for several swine-associated bacteria, including Streptococcus sp., Clostridium sp., and Staphylococcus sp.

Multiplex degenerate primer design for targeted whole genome amplification of many viral genomes.

Background. Targeted enrichment improves coverage of highly mutable viruses at low concentration in complex samples. Degenerate primers that anneal to conserved regions can facilitate amplification of divergent, low concentration variants, even when the strain present is unknown. Results. A tool for designing multiplex sets of degenerate sequencing primers to tile overlapping amplicons across multiple whole genomes is described. The new script, run_tiled_primers, is part of the PriMux software. Primers were designed for each segment of South American hemorrhagic fever viruses, tick-borne encephalitis, Henipaviruses, Arenaviruses, Filoviruses, Crimean-Congo hemorrhagic fever virus, Rift Valley fever virus, and Japanese encephalitis virus. Each group is highly diverse with as little as 5% genome consensus. Primer sets were computationally checked for nontarget cross reactions against the NCBI nucleotide sequence database. Primers for murine hepatitis virus were demonstrated in the lab to specifically amplify selected genes from a laboratory cultured strain that had undergone extensive passage in vitro and in vivo. Conclusions. This software should help researchers design multiplex sets of primers for targeted whole genome enrichment prior to sequencing to obtain better coverage of low titer, divergent viruses. Applications include viral discovery from a complex background and improved sensitivity and coverage of rapidly evolving strains or variants in a gene family.

Screening of viral pathogens from pediatric ileal tissue samples after vaccination.

In 2010, researchers reported that the two US-licensed rotavirus vaccines contained DNA or DNA fragments from porcine circovirus (PCV). Although PCV, a common virus among pigs, is not thought to cause illness in humans, these findings raised several safety concerns. In this study, we sought to determine whether viruses, including PCV, could be detected in ileal tissue samples of children vaccinated with one of the two rotavirus vaccines. A broad spectrum, novel DNA detection technology, the Lawrence Livermore Microbial Detection Array (LLMDA), was utilized, and confirmation of viral pathogens using the polymerase chain reaction (PCR) was conducted. The LLMDA technology was recently used to identify PCV from one rotavirus vaccine. Ileal tissue samples were analyzed from 21 subjects, aged 15-62 months. PCV was not detected in any ileal tissue samples by the LLMDA or PCR. LLMDA identified a human rotavirus A from one of the vaccinated subjects, which is likely due to a recent infection from a wild type rotavirus. LLMDA also identified human parechovirus, a common gastroenteritis viral infection, from two subjects. Additionally, LLMDA detected common gastrointestinal bacterial organisms from the Enterobacteriaceae, Bacteroidaceae, and Streptococcaceae families from several subjects. This study provides a survey of viral and bacterial pathogens from pediatric ileal samples, and may shed light on future studies to identify pathogen associations with pediatric vaccinations.

Bioinformatics for microbial genotyping of equine encephalitis viruses, orthopoxviruses, and hantaviruses.

Microbial genotyping is essential for forensic discrimination of pathogen strains, tracing epidemics, and understanding evolutionary processes. Phylogenetic analyses were performed and genotyping assays designed for five viral species complexes or genera: Western, Eastern, and Venezuelan equine encephalitis viruses, hantavirus segments L, M, and S, and orthopoxviruses. For each group, sequence alignments and phylogenetic trees were built. PCR signatures composed of primer pairs or TaqMan™ triplets were designed and mapped to nodes of the trees for sub-type or strain specific PCR-based identification. In addition, single nucleotide polymorphisms (SNPs) were identified and mapped to trees, and SNP microarray probes were designed to enable highly multiplexed genotyping of an unsequenced sample by hybridization. SNP-based trees corresponded well with MSA trees. Near-perfect isolate resolution was possible for all viruses analyzed computationally using either SNPs or PCR signatures. More tree nodes were represented by SNP loci than by PCR signatures, as PCR signatures often represented subsets of strains not corresponding to a branch. However, while PCR genotyping is possible, the number of PCR signatures needed to characterize an unknown can be very large. SNP microarrays are a suitable alternative, as arrays enable highly multiplexed, high resolution genotyping of an unknown in a single hybridization assay.