Cell fusing agent virus (Flavivirus) infection in Aedes aegypti in Texas: seasonality, comparison by trap type, and individual viral loads.

South Texas has experienced local transmission of Zika virus and of other mosquito-borne viruses such as chikungunya virus and dengue virus in the last decades. Using a mosquito surveillance program in the Lower Rio Grande Valley (LRGV) and San Antonio, TX, from 2016 to 2018, we detected the presence of an insect-specific virus, cell fusing agent virus (CFAV), in the Aedes aegypti mosquito population. We tested 6,326 females and 1,249 males from the LRGV and 659 females from San Antonio for CFAV by RT-PCR using specific primers. Infection rates varied from 0 to 261 per 1,000 mosquitoes in the LRGV and 115 to 208 per 1,000 in San Antonio depending on the month of collection. Infection rates per 1,000 individuals appeared higher in females collected from BG Sentinel 2 traps compared to Autocidal Gravid Ovitraps, but the ratio of the percentage of infected pools did not differ by trap type. The natural viral load in individual males ranged from 1.25 x 102 to 5.50 x 106 RNA copies and in unfed females from 5.42 x 103 to 8.70 x 106 RNA copies. Gravid females were found to harbor fewer viral particles than males and unfed females.

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.

Differential laboratory passaging of SARS-CoV-2 viral stocks impacts the in vitro assessment of neutralizing antibodies.

Viral populations in natural infections can have a high degree of sequence diversity, which can directly impact immune escape. However, antibody potency is often tested in vitro with a relatively clonal viral populations, such as laboratory virus or pseudotyped virus stocks, which may not accurately represent the genetic diversity of circulating viral genotypes. This can affect the validity of viral phenotype assays, such as antibody neutralization assays. To address this issue, we tested whether recombinant virus carrying SARS-CoV-2 spike (VSV-SARS-CoV-2-S) stocks could be made more genetically diverse by passage, and if a stock passaged under selective pressure was more capable of escaping monoclonal antibody (mAb) neutralization than unpassaged stock or than viral stock passaged without selective pressures. We passaged VSV-SARS-CoV-2-S four times concurrently in three cell lines and then six times with or without polyclonal antiserum selection pressure. All three of the monoclonal antibodies tested neutralized the viral population present in the unpassaged stock. The viral inoculum derived from serial passage without antiserum selection pressure was neutralized by two of the three mAbs. However, the viral inoculum derived from serial passage under antiserum selection pressure escaped neutralization by all three mAbs. Deep sequencing revealed the rapid acquisition of multiple mutations associated with antibody escape in the VSV-SARS-CoV-2-S that had been passaged in the presence of antiserum, including key mutations present in currently circulating Omicron subvariants. These data indicate that viral stock that was generated under polyclonal antiserum selection pressure better reflects the natural environment of the circulating virus and may yield more biologically relevant outcomes in phenotypic assays. Thus, mAb assessment assays that utilize a more genetically diverse, biologically relevant, virus stock may yield data that are relevant for prediction of mAb efficacy and for enhancing biosurveillance.

Ultra-deep mutant spectrum profiling: improving sequencing accuracy using overlapping read pairs.

BACKGROUND: High throughput sequencing is beginning to make a transformative impact in the area of viral evolution. Deep sequencing has the potential to reveal the mutant spectrum within a viral sample at high resolution, thus enabling the close examination of viral mutational dynamics both within- and between-hosts. The challenge however, is to accurately model the errors in the sequencing data and differentiate real viral mutations, particularly those that exist at low frequencies, from sequencing errors. RESULTS: We demonstrate that overlapping read pairs (ORP) -- generated by combining short fragment sequencing libraries and longer sequencing reads -- significantly reduce sequencing error rates and improve rare variant detection accuracy. Using this sequencing protocol and an error model optimized for variant detection, we are able to capture a large number of genetic mutations present within a viral population at ultra-low frequency levels (<0.05%). CONCLUSIONS: Our rare variant detection strategies have important implications beyond viral evolution and can be applied to any basic and clinical research area that requires the identification of rare mutations.

Multiple Mutations Associated with Emergent Variants Can Be Detected as Low-Frequency Mutations in Early SARS-CoV-2 Pandemic Clinical Samples.

Genetic analysis of intra-host viral populations provides unique insight into pre-emergent mutations that may contribute to the genotype of future variants. Clinical samples positive for SARS-CoV-2 collected in California during the first months of the pandemic were sequenced to define the dynamics of mutation emergence as the virus became established in the state. Deep sequencing of 90 nasopharyngeal samples showed that many mutations associated with the establishment of SARS-CoV-2 globally were present at varying frequencies in a majority of the samples, even those collected as the virus was first detected in the US. A subset of mutations that emerged months later in consensus sequences were detected as subconsensus members of intra-host populations. Spike mutations P681H, H655Y, and V1104L were detected prior to emergence in variant genotypes, mutations were detected at multiple positions within the furin cleavage site, and pre-emergent mutations were identified in the nucleocapsid and the envelope genes. Because many of the samples had a very high depth of coverage, a bioinformatics pipeline, "Mappgene", was established that uses both iVar and LoFreq variant calling to enable identification of very low-frequency variants. This enabled detection of a spike protein deletion present in many samples at low frequency and associated with a variant of concern.

Quantitative Fit Evaluation of N95 Filtering Facepiece Respirators and Coronavirus Inactivation Following Heat Treatment.

Reuse of filtering facepiece respirators (FFRs, commonly referred to as N95s) normally meant for single use has become common in healthcare facilities due to shortages caused by the COVID-19 pandemic. Here, we report that murine hepatitis coronavirus initially seeded on FFR filter material is inactivated (6 order of magnitude reduction as measured by median tissue culture infective dose, TCID50) after dry heating at 75°C for 30 min. We also find that the quantitative fit of FFRs after heat treatment at this temperature, under dry conditions or at 90% relative humidity, is not affected by single or 10 heating cycles. Previous studies have reported that the filtration efficiency of FFRs is not negatively impacted by these heating conditions. These results suggest that thermal inactivation of coronaviruses is a potentially rapid and widely deployable method to reuse N95 FFRs in emergency situations where reusing FFRs is a necessity and broad-spectrum sterilization is unavailable. However, we also observe that a radiative heat source (e.g. an exposed heating element) results in rapid qualitative degradation of the FFR. Finally, we discuss differences in the results reported here and other recent studies investigating heat as a means to recycle FFRs. These differences suggest that while our repeated decontamination cycles do not affect FFR fit, overall wear time and the number of donning/doffing cycles are important factors that likely degrade FFR fit and must be investigated further.

The Eco-Bio-Social Factors That Modulate Aedes aegypti Abundance in South Texas Border Communities.

Aedes aegypti control requires dedicated resources that are usually scarce, limiting the reach and sustainability of vector control programs. This generates a need to focus on areas at risk of disease transmission and also understand the factors that might modulate local mosquito abundance. We evaluated the eco-bio-social factors that modulate indoor and outdoor relative abundance of female Ae. aegypti in communities of South Texas. We conducted housing quality and Knowledge Attitudes and Practices surveys in households that were part of a weekly mosquito surveillance program in November of 2017 and 2018. Our results showed widespread knowledge of mosquitoes and Zika virus by our participants. However, less than 35% considered them as serious problems in this region. The presence of window-mounted air conditioning units increased the risk of female mosquito relative abundance indoors. An increase in outdoor relative abundance was associated with larger properties and a higher number of children between 6 to 17 years of age. Interestingly, we observed that an increasing number of children <5 years of age modulated both indoor and outdoor relative abundance, with a 52% increase indoors and 30% decrease outdoors. The low perception of mosquito and disease risk highlights engagement needs for vector-borne disease prevention in this region. The identified risk factors can help guide public health officials in their efforts to reduce human and vector contact.

Single Amino Acid Mutations Affect Zika Virus Replication In Vitro and Virulence In Vivo.

The 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence, and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks. To determine if naturally occurring individual mutations in the Zika virus epidemic genotype affect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

High Rate of Non-Human Feeding by Aedes aegypti Reduces Zika Virus Transmission in South Texas.

Mosquito-borne viruses are emerging or re-emerging globally, afflicting millions of people around the world. Aedes aegypti, the yellow fever mosquito, is the principal vector of dengue, Zika, and chikungunya viruses, and has well-established populations across tropical and subtropical urban areas of the Americas, including the southern United States. While intense arboviral epidemics have occurred in Mexico and further south in the Americas, local transmission in the United States has been minimal. Here, we study Ae. aegypti and Culex quinquefasciatus host feeding patterns and vertebrate host communities in residential environments of South Texas to identify host-utilization relative to availability. Only 31% of Ae. aegypti blood meals were derived from humans, while 50% were from dogs and 19% from other wild and domestic animals. In Cx. quinquefasciatus, 67% of blood meals were derived from chicken, 22% came from dogs, 9% from various wild avian species, and 2% from other mammals including one human, one cat, and one pig. We developed a model for the reproductive number, R0, for Zika virus (ZIKV) in South Texas relative to northern Mexico using human disease data from Tamaulipas, Mexico. We show that ZIKV R0 in South Texas communities could be greater than one if the risk of human exposure to Ae. aegypti bites in these communities is at least 60% that of Northern Mexico communities. The high utilization of non-human vertebrates and low risk of human exposure in South Texas diminishes the outbreak potential for human-amplified urban arboviruses transmitted by Ae. aegypti.

Multiscale analysis for patterns of Zika virus genotype emergence, spread, and consequence.

The question of how Zika virus (ZIKV) changed from a seemingly mild virus to a human pathogen capable of microcephaly and sexual transmission remains unanswered. The unexpected emergence of ZIKV's pathogenicity and capacity for sexual transmission may be due to genetic changes, and future changes in phenotype may continue to occur as the virus expands its geographic range. Alternatively, the sheer size of the 2015-16 epidemic may have brought attention to a pre-existing virulent ZIKV phenotype in a highly susceptible population. Thus, it is important to identify patterns of genetic change that may yield a better understanding of ZIKV emergence and evolution. However, because ZIKV has an RNA genome and a polymerase incapable of proofreading, it undergoes rapid mutation which makes it difficult to identify combinations of mutations associated with viral emergence. As next generation sequencing technology has allowed whole genome consensus and variant sequence data to be generated for numerous virus samples, the task of analyzing these genomes for patterns of mutation has become more complex. However, understanding which combinations of mutations spread widely and become established in new geographic regions versus those that disappear relatively quickly is essential for defining the trajectory of an ongoing epidemic. In this study, multiscale analysis of the wealth of genomic data generated over the course of the epidemic combined with in vivo laboratory data allowed trends in mutations and outbreak trajectory to be assessed. Mutations were detected throughout the genome via deep sequencing, and many variants appeared in multiple samples and in some cases become consensus. Similarly, amino acids that were previously consensus in pre-outbreak samples were detected as low frequency variants in epidemic strains. Protein structural models indicate that most of the mutations associated with the epidemic transmission occur on the exposed surface of viral proteins. At the macroscale level, consensus data was organized into large and interactive databases to allow the spread of individual mutations and combinations of mutations to be visualized and assessed for temporal and geographical patterns. Thus, the use of multiscale modeling for identifying mutations or combinations of mutations that impact epidemic transmission and phenotypic impact can aid the formation of hypotheses which can then be tested using reverse genetics.

Surveillance of Aedes aegypti indoors and outdoors using Autocidal Gravid Ovitraps in South Texas during local transmission of Zika virus, 2016 to 2018.

The yellow fever mosquito, Aedes aegypti, has facilitated the re-emergence of dengue virus (DENV) and emergence of chikungunya virus (CHIKV) and Zika virus (ZIKV) in the Americas and the Caribbean. The recent transmission of these arboviruses in the continental United States has been limited, to date, to South Florida and South Texas despite Ae. aegypti occurring over a much larger geographical region within the country. The main goal of our study was to provide the first long term longitudinal study of Ae. aegypti and enhance the knowledge about the indoor and outdoor relative abundance of Ae. aegypti as a proxy for mosquito-human contact in South Texas, a region of the United States that is at high risk for mosquito-borne virus transmission. Here, the relative abundance of indoors and outdoors mosquitoes of households in eight different communities was described. Surveillance was done weekly from September 2016 to April 2018 using the CDC Autocidal Gravid Ovitraps in low- and middle-income communities. A total of 69 houses were included in this survey among which 36 were in the low-income communities (n = 11 for Donna, n = 15 for Progresso, n = 5 for Mesquite, n = 5 for Chapa) and 33 in middle-income communities (n = 9 for La Feria, n = 8 for Weslaco, n = 11 for McAllen, and n = 5 for Rio Rico). Overall, Ae. aegypti was the dominant species (59.2% of collections, n = 7255) followed by Culex spp. mosquitoes (27.3% of collections, n = 3350). Furthermore, we demonstrated for Ae. aegypti that 1) outdoor relative abundance was higher compared to indoor relative abundance, 2) low-income communities were associated with an increase in mosquito relative abundance indoors when compared to middle-income communities, 3) no difference was observed in the number of mosquitoes collected outdoors between low-income and middle-income communities, and 4) warmer months were positively correlated with outdoor relative abundance whereas no seasonality was observed in the relative abundance of mosquitoes indoors. Additionally, Ae. aegypti mosquitoes collected in South Texas were tested using a specific ZIKV/CHIKV multiplex real-time PCR assay, however, none of the mosquitoes tested positive. Our data highlights the occurrence of mosquitoes indoors in the continental United States and that adults are collected nearly every week of the calendar year. These mosquito data, obtained concurrently with local ZIKV transmission of 10 locally acquired cases in nearby communities, represent a baseline for future studies in the Lower Rio Grande Valley (LRGV) including vector control interventions relying on the oviposition behavior to reduce mosquito populations and pathogen transmission.

Role of uvrA in the growth and survival of Listeria monocytogenes under UV radiation and acid and bile stress.

Listeria monocytogenes encounters numerous stresses both in the food environment and during infection of the host. The ability to survive and tolerate bile and low pH conditions, which are two major stresses, is of particular importance for survival within the host. The uvrA gene in other bacteria is involved in the repair of acid-induced DNA damage and in adaptation to low pH. Thus, a uvrA in-frame deletion mutant was constructed to identify the role of uvrA in the growth and survival of L. monocytogenes under various environmental conditions. The uvrA mutant was highly sensitive to UV radiation. Growth under normal laboratory conditions was impaired during the exponential phase, and the time to reach the exponential phase of growth, TV(max), was significantly delayed (P < 0.05). Growth of the uvrA mutant in acidic medium (pH 5) was slightly impaired, and the TV(max) was significantly delayed (P < 0.05). Growth and the TV(max) of the mutant in the presence of 0.3% bile salts also were significantly impaired (P < 0.05). These results suggest that uvrA is needed for optimal growth and survival of L. monocytogenes under various stressful environmental conditions.

Correction: Middle East Respiratory Syndrome Coronavirus Intra-Host Populations Are Characterized by Numerous High Frequency Variants.

[This corrects the article DOI: 10.1371/journal.pone.0146251.].

Middle East Respiratory Syndrome Coronavirus Intra-Host Populations Are Characterized by Numerous High Frequency Variants.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging human pathogen related to SARS virus. In vitro studies indicate this virus may have a broad host range suggesting an increased pandemic potential. Genetic and epidemiological evidence indicate camels serve as a reservoir for MERS virus but the mechanism of cross species transmission is unclear and many questions remain regarding the susceptibility of humans to infection. Deep sequencing data was obtained from the nasal samples of three camels that had been experimentally infected with a human MERS-CoV isolate. A majority of the genome was covered and average coverage was greater than 12,000x depth. Although only 5 mutations were detected in the consensus sequences, 473 intrahost single nucleotide variants were identified. Many of these variants were present at high frequencies and could potentially influence viral phenotype and the sensitivity of detection assays that target these regions for primer or probe binding.

Sendai virus intra-host population dynamics and host immunocompetence influence viral virulence during in vivo passage.

In vivo serial passage of non-pathogenic viruses has been shown to lead to increased viral virulence, and although the precise mechanism(s) are not clear, it is known that both host and viral factors are associated with increased pathogenicity. Under- or overnutrition leads to a decreased or dysregulated immune response and can increase viral mutant spectrum diversity and virulence. The objective of this study was to identify the role of viral mutant spectra dynamics and host immunocompetence in the development of pathogenicity during in vivo passage. Because the nutritional status of the host has been shown to affect the development of viral virulence, the diet of animal model reflected two extremes of diets which exist in the global population, malnutrition and obesity. Sendai virus was serially passaged in groups of mice with differing nutritional status followed by transmission of the passaged virus to a second host species, guinea pigs. Viral population dynamics were characterized using deep sequence analysis and computational modeling. Histopathology, viral titer and cytokine assays were used to characterize viral virulence. Viral virulence increased with passage and the virulent phenotype persisted upon passage to a second host species. Additionally, nutritional status of mice during passage influenced the phenotype. Sequencing revealed the presence of several non-synonymous changes in the consensus sequence associated with passage, a majority of which occurred in the hemagglutinin-neuraminidase and polymerase genes, as well as the presence of persistent high frequency variants in the viral population. In particular, an N1124D change in the consensus sequences of the polymerase gene was detected by passage 10 in a majority of the animals. In vivo comparison of an 1124D plaque isolate to a clone with 1124N genotype indicated that 1124D was associated with increased virulence.

La Crosse virus: replication in vertebrate and invertebrate hosts.

La Crosse virus is maintained in a cycle involving mosquitoes and small mammals. Vertebrate cell infection is generally cytolytic; vector cell infection results in persistent infection. Features of La Crosse virus replication that may permit the virus to traffic between vector and vertebrate hosts and condition different infection outcomes are described.

Complete nucleotide sequence of a Chilean hantavirus.

We have determined the genomic sequence of an Andes virus (ANDV) strain isolated from an infected Oligoryzomys longicaudatus rodent trapped in Chile in 1997. This strain, for which we propose the designation Chile R123, reproduces essential attributes of hantavirus pulmonary syndrome (HPS) when injected intramuscularly into laboratory hamsters (Hooper et al., Virology 289 (2001) 6-14). The L, M, and S segment sequences of Chile R123 are 6562, 3671, and 1871 nt long, respectively, with an overall G+C content of 38.5%. These respective genome segments could encode a 247 kd RNA-dependent RNA polymerase (RdRP), 126 kd glycoprotein precursor (GPC), and 48 kd nucleocapsid (N) protein, in line with other Sigmodontine rodent-associated hantaviruses. Among hantaviruses for which complete genomic sequences are available, Chile R123 is most closely related to Sin Nombre virus (SNV) strain NM R11, with greater than 85% amino acid identity between translated L and S segments and 78% amino acid identity between translated M segments. Because Chile R123 shares essentially 100% amino acid identity in regions of overlap with partially sequenced Argentinian and Chilean ANDV strains, Syrian hamster pathogenicity and the potential for interhuman transmission are features likely common to all ANDV strains.

Detection of bacterial pathogens in environmental samples using DNA microarrays.

Polymerase chain reaction (PCR) is an important tool for pathogen detection, but historically, it has not been possible to accurately identify PCR products without sequencing, Southern blots, or dot-blots. Microarrays can be coupled with PCR where they serve as a set of parallel dot-blots to enhance product detection and identification. Microarrays are composed of many discretely located probes on a solid substrate such as glass. Each probe is composed of a sequence that is complimentary to a pathogen-specific gene sequence. PCR is used to amplify one or more genes and the products are then hybridized to the array to identify species-specific polymorphism within one or more genes. We illustrate this type of array using 16S rDNA probes suitable for distinguishing between several salmonid pathogens. We also describe the use of microarrays for direct detection of either RNA or DNA without the aid of PCR, although the sensitivity of these systems currently limits their application for pathogen detection. Finally, microarrays can also be used to "fingerprint" bacterial isolates and they can be used to identify diagnostic markers suitable for developing new PCR-based detection assays. We illustrate this type of array for subtyping an important food-borne pathogen, Listeria monocytogenes.

Discrimination among Listeria monocytogenes isolates using a mixed genome DNA microarray.

Listeria monocytogenes can cause serious illness in humans, usually following the ingestion of contaminated food. Epidemiologic investigation requires identification of specific isolates, usually done by a combination of serotyping and subtyping using pulsed-field gel electrophoresis (PFGE). DNA microarrays provide a new format to resolve genetic differences among isolates and, unlike PFGE, to identify specific genes associated with the infecting pathogen. A 585 probe, mixed genome microarray was constructed and 24 strains of L. monocytogenes were hybridized to the array. Microarray analysis allowed discrimination among L. monocytogenes isolates within a serotype and obtained from similar geographic and epidemiologic sources. Importantly, the microarray results preserved previously described phylogenetic relationships between major serogroups and, in a limited comparison, agreed with PFGE subtypes. The association of individual probes with isolates allowed identification of specific genes. Sequencing of 10 polymorphic probes identified nine matches with previously described bacterial genes including several suspected virulence factors. These results demonstrate that mixed genomic microarrays are useful for differentiating among closely related L. monocytogenes isolates and identifying genetic markers that can be used in epidemiologic and possibly pathogenesis studies.

Dairy farm reservoir of Listeria monocytogenes sporadic and epidemic strains.

Identifying the reservoirs of a pathogen is vital for control of sporadic disease and epidemics. Listeria monocytogenes is a zoonotic foodborne pathogen that is responsible for 28% of food-related deaths in the United States annually, as well as a major cause of massive product recalls worldwide. To examine the role of the dairy farm as a potential source or reservoir for L. monocytogenes subtypes shown to cause human listeriosis, we compared the pulsed-field gel electrophoresis (PFGE) restriction enzyme digestion profiles of L. monocytogenes dairy farm-associated strains (milk, environmental, and bovine) to human sporadic and epidemic disease strains. Twenty-three percent of human sporadic strains had PFGE patterns identical to that of farm isolate(s). Additionally, three farm environmental strains and one human sporadic strain had a PFGE pattern identical to a strain of L. monocytogenes responsible for the 1985 California epidemic. These data indicate that this epidemic strain continues to cause sporadic human illness and has a potential dairy farm as a reservoir.