[show abstract][hide abstract] ABSTRACT: Thirty-nine human parainfluenza type 1 (HPIV-1) genomes were sequenced from samples collected in Milwaukee, Wisconsin from 1997-2010. Following sequencing, phylogenetic analyses of these sequences plus any publicly available HPIV-1 sequences (from GenBank) were performed. Phylogenetic analysis of the whole genomes, as well as individual genes, revealed that the current HPIV-1 viruses group into three different clades. Previous evolutionary studies of HPIV-1 in Milwaukee revealed that there were two genotypes of HPIV-1 co-circulating in 1991 (previously described as HPIV-1 genotypes C and D). The current study reveals that there are still two different HPIV-1 viruses co-circulating in Milwaukee; however, both groups of HPIV-1 viruses are derived from genotype C indicating that genotype D may no longer be in circulation in Milwaukee. Analyses of genetic diversity indicate that while most of the genome is under purifying selection some regions of the genome are more tolerant of mutation. In the 40 HPIV-1 genomes sequenced in this study, the nucleotide sequence of the L gene is the most conserved while the sequence of the P gene is the most variable. Over the entire protein coding region of the genome, 81 variable amino acid residues were observed and as with nucleotide diversity, the P protein seemed to be the most tolerant of mutation (and contains the greatest proportion of non-synonymous to synonymous substitutions) while the M protein appears to be the least tolerant of amino acid substitution.
PLoS ONE 01/2012; 7(9):e46048. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Respiratory Syncytial Virus (RSV) is the leading cause of lower respiratory-tract infections in infants and young children worldwide. Despite this, only six complete genome sequences of original strains have been previously published, the most recent of which dates back 35 and 26 years for RSV group A and group B respectively.
We present a semi-automated sequencing method allowing for the sequencing of four RSV whole genomes simultaneously. We were able to sequence the complete coding sequences of 13 RSV A and 4 RSV B strains from Milwaukee collected from 1998-2010. Another 12 RSV A and 5 RSV B strains sequenced in this study cover the majority of the genome. All RSV A and RSV B sequences were analyzed by neighbor-joining, maximum parsimony and Bayesian phylogeny methods. Genetic diversity was high among RSV A viruses in Milwaukee including the circulation of multiple genotypes (GA1, GA2, GA5, GA7) with GA2 persisting throughout the 13 years of the study. However, RSV B genomes showed little variation with all belonging to the BA genotype. For RSV A, the same evolutionary patterns and clades were seen consistently across the whole genome including all intergenic, coding, and non-coding regions sequences.
The sequencing strategy presented in this work allows for RSV A and B genomes to be sequenced simultaneously in two working days and with a low cost. We have significantly increased the amount of genomic data that is available for both RSV A and B, providing the basic molecular characteristics of RSV strains circulating in Milwaukee over the last 13 years. This information can be used for comparative analysis with strains circulating in other communities around the world which should also help with the development of new strategies for control of RSV, specifically vaccine development and improvement of RSV diagnostics.
PLoS ONE 01/2011; 6(10):e25468. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spatial variation in the epidemiological patterns of successive waves of pandemic influenza virus in humans has been documented throughout the 20th century but never understood at a molecular level. However, the unprecedented intensity of sampling and whole-genome sequencing of the H1N1/09 pandemic virus now makes such an approach possible. To determine whether the spring and fall waves of the H1N1/09 influenza pandemic were associated with different epidemiological patterns, we undertook a large-scale phylogeographic analysis of viruses sampled from three localities in the United States. Analysis of genomic and epidemiological data reveals distinct spatial heterogeneities associated with the first pandemic wave, March to July 2009, in Houston, TX, Milwaukee, WI, and New York State. In Houston, no specific H1N1/09 viral lineage dominated during the spring of 2009, a period when little epidemiological activity was observed in Texas. In contrast, major pandemic outbreaks occurred at this time in Milwaukee and New York State, each dominated by a different viral lineage and resulting from strong founder effects. During the second pandemic wave, beginning in August 2009, all three U.S. localities were dominated by a single viral lineage, that which had been dominant in New York during wave 1. Hence, during this second phase of the pandemic, extensive viral migration and mixing diffused the spatially defined population structure that had characterized wave 1, amplifying the one viral lineage that had dominated early on in one of the world's largest international travel centers.
Journal of Virology 11/2010; 85(2):828-34. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Respiratory tract viral infections are responsible for an incredible amount of morbidity and mortality throughout the world. Older diagnostic methods, such as tissue culture and serology, have been replaced with more advanced molecular techniques, such as PCR and reverse-transcriptase PCR, nucleic acid sequence-based amplification and loop-mediated isothermal amplification. These techniques are faster, have greater sensitivity and specificity, and are becoming increasingly accessible. In the minds of most, PCR has replaced tissue culture and serology as the gold standard for detection of respiratory viruses owing to its speed, availability and versatility. PCR/reverse-transcriptase PCR has been used in a variety of detection platforms, in multiplex assays (detecting multiple pathogens simultaneously) and in automated systems (sample in-answer out devices). Molecular detection has many proven advantages over standard virological methods and will further separate itself through increased multiplexing, processing speed and automation. However, tissue culture remains an important method for detecting novel viral mutations within a virus population, for detecting novel viruses and for phenotypic characterization of viral isolates.
[show abstract][hide abstract] ABSTRACT: Polymerase chain reaction (PCR) assays increase the rate of viral detection in clinical specimens, compared with conventional virologic methods. Studies suggest that PCR may detect virus nucleic acid (NA) that persists in the respiratory tract.
We analyzed virologic data from children having frequent upper respiratory infections (URI), who were followed up in a longitudinal study. Nasopharyngeal secretions were collected at URI onset and when acute otitis media was diagnosed; virus studies were performed using conventional diagnostics and PCR. Repeated presence of adenovirus by PCR was further studied by sequencing and phylogenetic analysis.
Of 581 URI episodes in 76 children, 510 viruses were detected. Of the viruses detected by PCR, 15% were those detected previously; repeated positives occurred most frequently with adenovirus. Sequencing results were available in 13 children with repeated adenovirus detection; the following 4 patterns of infection were identified (16 instances): (1) adenovirus of the same serotype and strain detected continuously (n = 8 instances), (2) adenovirus of different serotypes detected during sequential URI episodes (n = 3), (3) adenovirus of the same serotype but different strains detected during sequential URI episodes (n = 3), and (4) adenovirus of the same serotype and strain detected intermittently (n = 2).
Among children with frequent URIs, repeated positive PCR results for adenovirus NA may represent a new serotype/strain, or persistence of viral NA. Results must be interpreted with caution; clinical correlation and presence of other viruses are important. Further longitudinal studies of children during and after infection are required for better understanding of the clinical significance of positive PCR tests for adenovirus NA in the respiratory tract.
[show abstract][hide abstract] ABSTRACT: La Crosse virus (LACV) is a pathogenic arbovirus that is transovarially transmitted by Aedes triseriatus mosquitoes and overwinters in diapausing eggs. However, previous models predicted transovarial transmission (TOT) to be insufficient to maintain LACV in nature.
To investigate this issue, we reared mosquitoes from field-collected eggs and assayed adults individually for LACV antigen, viral RNA by RT-PCR, and infectious virus. The mosquitoes had three distinct infection phenotypes: 1) super infected (SI+) mosquitoes contained infectious virus, large accumulations of viral antigen and RNA and comprised 17 of 17,825 (0.09%) of assayed mosquitoes, 2) infected mosquitoes (I+) contained no detectable infectious virus, lesser amounts of viral antigen and RNA, and comprised 3.7% of mosquitoes, and 3) non-infected mosquitoes (I-) contained no detectable viral antigen, RNA, or infectious virus and comprised 96.21% of mosquitoes. SI+ mosquitoes were recovered in consecutive years at one field site, suggesting that lineages of TOT stably-infected and geographically isolated Ae. triseriatus exist in nature. Analyses of LACV genomes showed that SI+ isolates are not monophyletic nor phylogenetically distinct and that synonymous substitution rates exceed replacement rates in all genes and isolates. Analysis of singleton versus shared mutations (Fu and Li's F*) revealed that the SI+ LACV M segment, with a large and significant excess of intermediate-frequency alleles, evolves through disruptive selection that maintains SI+ alleles at higher frequencies than the average mutation rate. A QTN in the LACV NSm gene was detected in SI+ mosquitoes, but not in I+ mosquitoes. Four amino acid changes were detected in the LACV NSm gene from SI+ but not I+ mosquitoes from one site, and may condition vector super infection. In contrast to NSm, the NSs sequences of LACV from SI+ and I+ mosquitoes were identical.
SI+ mosquitoes may represent stabilized infections of Ae. triseriatus mosquitoes, which could maintain LACV in nature. A gene-for-gene interaction involving the viral NSm gene and a vector innate immune response gene may condition stabilized infection.
[show abstract][hide abstract] ABSTRACT: Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.
The Journal of molecular diagnostics: JMD 12/2009; 12(1):74-81. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: A large outbreak of novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) infection in Milwaukee, WI, occurred in late April 2009. We had recently developed a rapid multiplex reverse transcription-PCR enzyme hybridization assay (FluPlex) to determine the type (A or B) and subtype (H1, H2, H3, H5, H7, H9, N1 [human], N1 [animal], N2, or N7) of influenza viruses, and this assay was used to confirm the diagnoses for the first infected patients in the state. The analytical sensitivity was excellent at 1.5 to 116 copies/reaction, or 10(-3) to 10(-1) 50% tissue culture infective doses/ml. The testing of all existing hemagglutinin and neuraminidase subtypes of influenza A virus and influenza B virus (41 influenza virus strains) and 24 common respiratory pathogens showed only one low-level H3 cross-reaction with an H10N7 avian strain and only at 5.2 x 10(6) copies/reaction, not at lower concentrations. Comparisons of the FluPlex results with results from multiple validated in-house molecular assays, CDC-validated FDA-approved assays, and gene sequencing demonstrated 100% positive agreement for the typing of 179 influenza A viruses and 3 influenza B viruses, the subtyping of 110 H1N1 (S-OIV; N1 [animal]), 62 H1N1 (human), and 6 H3N2 (human) viruses, and the identification of 24 negative clinical samples and 100% negative agreement for all viruses tested except H1N1 (human) (97.7%). The small number of false-positive H1N1 (human) samples most likely represent increased sensitivity over that of other in-house assays, with four of four results confirmed by the CDC's influenza virus subtyping assay. The FluPlex is a rapid, inexpensive, sensitive, and specific method for the typing and subtyping of influenza viruses and demonstrated outstanding utility during the first 2 weeks of an S-OIV infection outbreak. Methods for rapid detection and broad subtyping of influenza viruses, including animal subtypes, are needed to address public concern over the emergence of pandemic strains. Attempts to automate this assay are ongoing.
Journal of clinical microbiology 08/2009; 47(9):2772-8. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the spring of 2009, a novel influenza A (H1N1) virus (swine origin influenza virus [S-OIV]) emerged and began causing a large outbreak of illness in Milwaukee, WI. Our group at the Midwest Respiratory Virus Program laboratory developed a semiautomated real-time multiplex reverse transcription-PCR assay (Seasonal), employing the NucliSENS easyMAG system (bioMérieux, Durham, NC) and a Raider thermocycler (HandyLab Inc., Ann Arbor, MI), that typed influenza A virus, influenza B virus, and respiratory syncytial virus (RSV) and subtyped influenza A virus into the currently circulating H1 and H3 subtypes, as well as a similar assay that identified H1 of S-OIV. The Seasonal and H1 S-OIV assays demonstrated analytical limits of detection of <50 50% tissue culture infective doses/ml and 3 to 30 input copies, respectively. Testing of the analytical specificities revealed no cross-reactivity with 41 and 26 different common organisms and demonstrated outstanding reproducibility of results. Clinical testing showed 95% sensitivity for influenza A virus and influenza B virus and 95 and 97% specificity compared to tissue culture. Comparisons of results from other molecular tests showed levels of positive agreement with the Seasonal and H1 S-OIV assay results of 99 and 100% and levels of negative agreement of 98 and 100%. This study has demonstrated the use of a semiautomated system for sensitive, specific, and rapid detection of influenza A virus, influenza B virus, and RSV and subtyping of influenza A virus into human H1 and H3 and S-OIV strains. This assay/system performed well in clinical testing of regular seasonal influenza virus subtypes and was outstanding during the 2009 Milwaukee S-OIV infection outbreak. This recent outbreak of infection with a novel influenza A (H1N1) virus also demonstrates the importance of quickly distributing information on new agents and of having rapid influenza virus subtyping assays widely available for clinical and public health decisions.
Journal of clinical microbiology 07/2009; 47(9):2779-86. · 4.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aedes triseriatus mosquitoes transovarially transmit (TOT) La Crosse virus (LACV) to their offspring with minimal damage to infected ovaries. Ae. triseriatus inhibitor of apoptosis 1 (AtIAP1) is a candidate gene conditioning the ability to vertically transmit LACV. AtIAP1 was amplified and sequenced in adult mosquitoes reared from field-collected eggs. Sequence analysis showed that AtIAP1 has much higher levels of genetic diversity than genes found in other mosquitoes. Despite this large amount of diversity, strong purifying selection of polymorphisms located in the Baculovirus inhibitor of apoptosis repeat (BIR) domains and, to a lesser extent, in the 5' untranslated region seems to indicate that these portions of AtIAP1 are the most important. These results indicate that the 5'UTR plays an important role in transcription and translation and that the BIR domains are important functional domains in the protein. Single nucleotide polymorphisms (SNPs) were compared between LACV-positive and -negative mosquitoes to test for associations between segregating sites and the ability to be transovarially infected with LACV. Initial results indicated that five SNPs were associated with TOT of LACV; however, these results were not replicable with larger sample sizes.
Journal of Molecular Evolution 04/2009; 68(4):403-13. · 2.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: Background Since its initial detection in April 2009, the A/H1N1pdm influenza virus has spread rapidly in humans, with over 5,700 human deaths. However, little is known about the evolutionary dynamics of H1N1pdm and its geographic and temporal diversification.Methods Phylogenetic analysis was conducted upon the concatenated coding regions of whole-genome sequences from 290 H1N1pdm isolates sampled globally between April 1 - July 9, 2009, including relatively large samples from the US states of Wisconsin and New York. Results At least 7 phylogenetically distinct viral clades have disseminated globally and co-circulated in localities that experienced multiple introductions of H1N1pdm. The epidemics in New York and Wisconsin were dominated by two different clades, both phylogenetically distinct from the viruses first identified in California and Mexico, suggesting an important role for founder effects in determining local viral population structures. Conclusions Determining the global diversity of H1N1pdm is central to understanding the evolution and spatial spread of the current pandemic, and to predict its future impact on human populations. Our results indicate that H1N1pdm has already diversified into distinct viral lineages with defined spatial patterns.
[show abstract][hide abstract] ABSTRACT: We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.
[show abstract][hide abstract] ABSTRACT: A population genetics study of the mosquito Ochlerotatus triseriatus was performed on 36 collections from adjoining regions of Iowa, Minnesota, and Wisconsin covering approximately 120 km(2). Single nucleotide polymorphism analysis was used to estimate variation in the mitochondrial NADH dehydrogenase subunit 4 (ND4) gene. The heated oligonucleotide ligation assay was used to identify the ND4 haplotype of each mosquito. No evidence of genetic isolation by distance was found, nor did Interstate 90 or the Mississippi River serve as barriers to gene flow. The effective migration rate varied from 18 to 45 reproductive migrants/generation, which is similar to estimates from an earlier study. The collections belong to a single, large, panmictic population. However, within this panmictic population, local genetic drift arises, possibly due to one or a few females ovipositing in larval breeding containers. From generation to generation, there is sufficient gene flow to mix families arising from individual breeding sites and eliminate founder effects due to drift.
The American journal of tropical medicine and hygiene 10/2005; 73(3):534-40. · 2.53 Impact Factor