Factors associated with peridomestic Triatoma sanguisuga (Hemiptera: Reduviidae) presence in southeastern Louisiana.

Although rare, there have been isolated reports of autochthonous transmission of Trypanosoma cruzi Chagas in the United States. In June 2006, a human case of domestically transmitted T. cruzi was identified in southern Louisiana. To examine the localized risk of human T. cruzi infection in the area surrounding the initial human case, environmental surveys of households in the area and a serological survey of the residents were performed between September 2008 and November 2009. Human T. cruzi infection was determined using a rapid antigen field test, followed by confirmatory enzyme-linked immunosorbent assay testing in the laboratory. A perimeter search of each participating residence for Triatoma sanguisuga (LeConte), the predominant local triatomine species, was also performed. No participating individuals were positive for antibodies against T. cruzi; however, high levels of T. cruzi infection (62.4%) were detected in collected T. sanguisuga. Households with T. sanguisuga presence were less likely to use air conditioning, and more likely to have either chickens or cats on the property. While the human risk for T cruzi infection in southeastern Louisiana is low, a high prevalence of infected T. sanguisuga does indicate a substantial latent risk for T. cruzi peridomestic transmission. Further examination of the behavior and ecology of T. sanguisuga in the region will assist in refining local T. cruzi risk associations.

Temperature, viral genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes.

The distribution and intensity of transmission of vector-borne pathogens can be strongly influenced by the competence of vectors. Vector competence, in turn, can be influenced by temperature and viral genetics. West Nile virus (WNV) was introduced into the United States of America in 1999 and subsequently spread throughout much of the Americas. Previously, we have shown that a novel genotype of WNV, WN02, first detected in 2001, spread across the US and was more efficient than the introduced genotype, NY99, at infecting, disseminating, and being transmitted by Culex mosquitoes. In the current study, we determined the relationship between temperature and time since feeding on the probability of transmitting each genotype of WNV. We found that the advantage of the WN02 genotype increases with the product of time and temperature. Thus, warmer temperatures would have facilitated the invasion of the WN02 genotype. In addition, we found that transmission of WNV accelerated sharply with increasing temperature, T, (best fit by a function of T(4)) showing that traditional degree-day models underestimate the impact of temperature on WNV transmission. This laboratory study suggests that both viral evolution and temperature help shape the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on WNV transmission.

A newly emergent genotype of West Nile virus is transmitted earlier and more efficiently by Culex mosquitoes.

Studies examining the evolution of West Nile virus since its introduction into North America have identified the emergence of a new dominant genotype (WN02) that has displaced the introduced genotype (NY99). The mechanistic basis for this displacement, however, remains obscure. Although we found no detectable difference in vitro between the genotypes in either replication or fitness, there were significant differences in vivo in Culex mosquitoes. After peroral infection, the extrinsic incubation period (EIP) of the WN02 genotype was up to 4 days shorter than the EIP of the NY99 genotype; however, after intrathoracic inoculation, there was no difference in EIP between the genotypes, suggesting that differences in genotype interaction with the mosquito midgut are likely to play a role in this phenotype. These results suggest a model for the displacement of the NY99 genotype, where earlier transmission of WN02 viruses leads to higher WN02 infection rates in avian reservoir hosts.

Antigenic and genetic variation in human respiratory syncytial virus.

BACKGROUND: Human respiratory syncytial virus (HRSV) is a leading cause of serious pediatric respiratory disease worldwide. Natural infection provides only partial protection as repeat infections occur throughout life. A brief review of the extent of antigenic and genetic variation observed in HRSV clinical isolates is presented. METHODS AND RESULTS: Recent experimental research is reviewed, describing key factors that may explain the ability of HRSV to cause multiple infections in the same individual even in the presence of an existing immune response. It is well-appreciated that variability of the G protein, both between and within antigenic subgroups A and B, is partially responsible for repeat HRSV infections. A high level of nucleotide change resulting in amino acid change provides strong evidence for selective pressure for change in G sequences, thus new HRSV variants. Although little variation in gene-coding sequences is observed in the F protein (the second major protective antigen), new evidence of genetic variation has identified alteration of gene expression levels by selection of changes in the gene end termination signal that precedes the gene encoding the F protein. Due to obligatory sequential transcription, these changes affect downstream gene expression levels. These data suggest that modulation of F protein levels may provide a selective advantage in the presence of a preexisting immune response. CONCLUSIONS: Experimental data in HRSV demonstrate that variation exists not only in gene-coding sequences but also in the signals that control gene expression. Thus alteration in the expression of key proteins provides a second type of antigenic "variation." A better understanding of these differences is critical to the development of an effective vaccine.

Bridging the health security divide: department of defense support for the global health security agenda.

In 2011, President Obama addressed the United Nations General Assembly and urged the global community to come together to prevent, detect, and fight every kind of biological danger, whether a pandemic, terrorist threat, or treatable disease. Over the past decade, the United States and key international partners have addressed these dangers through a variety of programs and strategies aimed at developing and enhancing countries' capacity to rapidly detect, assess, report, and respond to acute biological threats. Despite our collective efforts, however, an increasingly interconnected world presents heightened opportunities for human, animal, and zoonotic diseases to emerge and spread globally. Further, the technical capabilities required to develop biological agents into a weapon are relatively low. The launch of the Global Health Security Agenda (GHSA) provides an opportunity for the international community to enhance the linkages between the health and security sectors, accelerating global efforts to prevent avoidable epidemics and bioterrorism, detect threats early, and respond rapidly and effectively to biological threats. The US Department of Defense (DoD) plays a key role in achieving GHSA objectives through its force health protection, threat reduction, and biodefense efforts at home and abroad. This article focuses on GHSA activities conducted in the DoD Office of the Assistant Secretary of Defense for Nuclear, Chemical, and Biological Defense.

Requirement of glycosylation of West Nile virus envelope protein for infection of, but not spread within, Culex quinquefasciatus mosquito vectors.

Most of sequenced West Nile virus (WNV) genomes encode a single N-linked glycosylation site on their envelope (E) proteins. We previously found that WNV lacking the E protein glycan was severely inhibited in its ability to replicate and spread within two important mosquito vector species, Culex pipiens and Cx. tarsalis. However, recent work with a closely related species, Cx. pipiens pallens, found no association between E protein glycosylation and either replication or dissemination. To examine this finding further, we expanded upon our previous studies to include an additional Culex species, Cx. quinquefasciatus. The non-glycosylated WNV-N154I virus replicated less efficiently in mosquito tissues after intrathoracic inoculation, but there was little difference in replication efficiency in the midgut after peroral infection. Interestingly, although infectivity was inhibited when WNV lacked the E protein glycan, there was little difference in viral spread throughout the mosquito. These data indicate that E protein glycosylation affects WNV-vector interactions in a species-specific manner.

West Nile virus envelope protein glycosylation is required for efficient viral transmission by Culex vectors.

Many, but not all, strains of West Nile virus (WNV) contain a single N-linked glycosylation site on their envelope (E) proteins. Previous studies have shown that E-glycosylated strains are more neuroinvasive in mice than non-glycosylated strains. E protein glycosylation also appears to play a role in attachment and entry of WNV into host cells in vitro; however, studies examining how E protein glycosylation affects the interactions of WNV with its mosquito vectors in vivo have not yet been performed. We mutated the E protein glycosylation site from NYS to IYS in a previously described full-length clone of the NY99 genotype of WNV (WT), resulting in a virus that lacked the glycan at aa154. WNV-N154I replicated less efficiently than WNV-WT in Culex mosquito tissues, although the extent of the decrease was greater in Cx. pipiens than in Cx. tarsalis. Following peroral infection, mosquitoes infected with WNV-N154I were less likely to transmit virus than those infected with WNV-WT. Interestingly, all but one of the mosquitoes infected with WNV-N154I transmitted a revertant virus, suggesting that there is strong selective pressure toward E protein glycosylation. Together these data suggest that loss of the glycan at aa154 on the WNV E protein can severely restrict viral spread in the mosquito vector.

Characterization of a small plaque variant of West Nile virus isolated in New York in 2000.

A small-plaque variant (SP) of West Nile virus (WNV) was isolated in Vero cell culture from kidney tissue of an American crow collected in New York in 2000. The in vitro growth of the SP and parental (WT) strains was characterized in mammalian (Vero), avian (DF-1 and PDE), and mosquito (C6/36) cells. The SP variant replicated less efficiently than did the WT in Vero cells. In avian cells, SP growth was severely restricted at high temperatures, suggesting that the variant is temperature sensitive. In mosquito cells, growth of SP and WT was similar, but in vivo in Culex pipiens (L.) there were substantial differences. Relative to WT, SP exhibited reduced replication following intrathoracic inoculation and lower infection, dissemination, and transmission rates following oral infection. Analysis of the full length sequence of the SP variant identified sequence differences which led to only two amino acid substitutions relative to WT, prM P54S and NS2A V61A.

Variations in intergenic region sequences of Human respiratory syncytial virus clinical isolates: analysis of effects on transcriptional regulation.

Sequences at the beginnings and ends of Human respiratory syncytial virus (HRSV) genes are necessary for efficient initiation and termination of transcription. The gene start sequences are well conserved and contain signals required for initiation, while the semi-conserved sequences at the gene ends direct transcriptional termination with varying efficiencies. The intergenic regions, which lie between the gene ends and the downstream gene start sequences, are not conserved in length or sequence, and certain positions have been reported to play a role in transcriptional regulation. We have previously shown that the gene end sequences in HRSV subgroup A clinical isolates are variable and that variations found at certain gene ends decreased transcriptional termination and downstream mRNA expression. Here, we have extended this work to examine variation in the intergenic regions between the genes of clinical isolates. We determined the sequences of the eight intergenic regions and the M2/L overlap from clinical isolates from the US and UK and found that all of these regions contained variations from the prototype A2 strain. The amount of variation observed was disparate among the different intergenic regions and did not correlate with length. The effects of selected variant sequences on transcription were examined in the context of subgenomic replicons. While some changes in the intergenic regions had minor effects, certain sequence variations significantly altered transcription termination or initiation. A single nucleotide deletion in the M/SH intergenic region decreased initiation at the SH gene start seven-fold, while changes in the F/M2 intergenic region were found that in some cases increased and in others decreased termination at the F gene end. The P/M intergenic region was the most variable, but none of the changes examined affected either termination at the P gene end or initiation of the downstream M gene start. These results show that in HRSV clinical isolates the intergenic region sequences are variable and that changes in these regions have the potential to affect transcriptional control at the gene junctions.

Variations in transcription termination signals of human respiratory syncytial virus clinical isolates affect gene expression.

Human respiratory syncytial virus (HRSV) has a single-stranded, negative-sense RNA genome with 10 genes encoding 11 proteins. Sequences at the beginning of the HRSV genes are highly conserved; however, the gene end sequences vary around a semiconserved consensus sequence, and the nontranscribed intergenic regions vary in both length and sequence. The regions at the junctions between HRSV genes (the gene end sequence of an upstream gene, intergenic region, and the gene start sequence of a downstream gene) contain elements required for efficient termination of the upstream gene and transcription of the downstream gene. Previous studies have examined variation in the HRSV coding sequences, but none have systematically analyzed the noncoding transcriptional control regions for variability. We determined the gene start and gene end sequences of each of the 10 HRSV genes from 14 clinical isolates for variations from the sequence of the prototype A2 strain. No changes were found in any of the gene start sequences. Eight of the 10 gene end sequences, however, contained variations. Several of these, a U(4)-tract instead of a U(6)- or U(5)-tract at the M and SH gene ends, respectively, (U(4)A) and an A-to-G change at position four in the G gene end (A4G), were predicted to affect termination and were examined for their effects on transcription. The changes were found to inhibit transcriptional termination, resulting in increased polycistronic readthrough and correspondingly reduced initiation of the downstream monocistronic mRNA. Viruses with the A4G variant G gene end sequence produced less F protein than those with A2-like G gene end sequences. Examination of additional G gene end sequences available in GenBank revealed that the observed A4G variation was restricted to one phylogenetic lineage of HRSV. All viruses examined within this lineage possessed this variant G gene end sequence. The data presented show that the gene end sequences of naturally occurring HRSV clinical isolates vary from those of the prototypic A2 strain and that certain of these changes inhibit efficient transcriptional termination and downstream gene expression.