Associations between Wolbachia, maize and Diabrotica virgifera virgifera

Title from PDF of title page (University of Missouri--Columbia, viewed on Feb. 24, 2010).The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Dissertation advisor: Dr. Georgia Davis.VitaPh. D. University of Missouri--Columbia 2008.Diabrotica virgifera virgifera which is infected with Wolbachia, is the most significant and widespread pest to maize in North America and Europe. Wolbachia are obligate intracellular bacteria which seem to be limited to ecdyzoan hosts. Many Wolbachia hosts induce or vector serious human diseases resulting in the loss of millions of lives annually. The majority of differentially expressed genes identified in a microarray experiment performed to locate endogenous sources of D. virgifera virgifera resistance in maize, Zea mays L., are normally involved in microbial defense rather than wounding or insect attack. A second microarray experiment to determine whether Wolbachia were influencing the response of maize to D. virgifera virgifera attack indicated the presence of Wolbachia in the insect can down regulate genes in all plant defense classes. This may contribute to the success of D. virgifera virgifera as a maize pest. To further test this idea, assays on D. virgifera virgifera larval competitiveness and fertility were performed. Results of the assay suggest D. virgifera virgifera and Wolbachia share a commensal association.Includes bibliographical reference

Evidence of chikungunya virus disease in Pakistan since 2015 with patients demonstrating involvement of the central nervous system

Several arboviruses are endemic to and co-circulate in Pakistan. In recent years, Pakistan has observed a rise in arboviral infections. A cross-sectional study for arboviral diseases, which included screening for Chikungunya virus (CHIKV), was initiated in 2015 to determine which pathogens were causing disease in patients presenting to health care services. Exposure to CHIKV was verified via detection of viral nucleic acids or virus-specific IgM with virus-specific neutralizing antibodies. Out of 997 enrolled patients presenting with clinical features suggestive of arboviral disease, 102 patients were positive for CHIKV IgM antibodies and 60 patients were positive for CHIKV nucleic acids or neutralizing antibodies. The data presented here show that CHIKV has been circulating in Pakistan since April of 2015. CHIKV infections were detected in study subjects up to the conclusion of our enrollment period in July 2017. Syndromic and clinical data show that arthralgia was associated with CHIKV as was rash, fever greater than 38°C, and lymphopenia. Neurological symptoms were reported in 49% of CHIKV suspect patients and in 46.6% of confirmed infections. Acute disseminated encephalomyelitis was diagnosed in 5% of confirmed infection and various manifestation of encephalitis diagnosed in an additional 16.6% of patients with confirmed CHIKV infections. CHIKV-exposed patients were just as likely to present with neurological symptoms and encephalitis as patients with West Nile Virus infections but were 4.57 times more likely to have lymphopenia. This proportion of neurological symptoms may be a complicating factor in countries where WNV and/or JEV co-circulate with CHIKV

Comparison of clinical presentation and out-comes of chikungunya and dengue virus infections in patients with acute undifferentiated febrile illness from the Sindh region of Pakistan

Background: Arboviruses are a cause of acute febrile illness and outbreaks worldwide. Recent outbreaks of Chikungunya virus (CHIKV) in dengue endemic areas have alarmed clinicians as unique clinical features differentiating CHIKV from Dengue virus (DENV) are limited. This has complicated diagnostic efforts especially in resource limited countries where lab testing is not easily available. Therefore, it is essential to analyse and compare clinical features of laboratory confirmed cases to assist clinicians in suspecting possible CHIKV infection at time of clinical presentation.Methodology: A prospective point prevalence study was conducted, with the hypothesis that not all patients presenting with clinical suspicion of dengue infections at local hospitals are suffering from dengue and that other arboviruses such as Chikungunya, West Nile viruses, Japanese Encephalitis virus and Zika virus are co-circulating in the Sindh region of Pakistan. Out-patients and hospitalized (in-patients) of selected district hospitals in different parts of Sindh province of Pakistan were recruited. Patients with presumptive dengue like illness (Syndromic diagnosis) by the treating physicians were enrolled between 2015 and 2017. Current study is a subset of larger study mentioned above. Here-in we compared laboratory confirmed cases of CHIKV and DENV to assess clinical features and laboratory findings that may help differentiate CHIKV from DENV infection at the time of clinical presentation.Results: Ninety-eight (n = 98) cases tested positive for CHIKV, by IgM and PCR and these were selected for comparative analysis with DENV confirmed cases (n = 171). On multivariable analysis, presence of musculoskeletal [OR = 2.5 (95% CI:1.6-4.0)] and neurological symptoms [OR = 4.4 (95% CI:1.9-10.2)], and thrombocytosis [OR = 2.2 (95% CI:1.1-4.0)] were associated with CHIKV infection, while atypical lymphocytes [OR = 8.3 (95% CI:4.2-16.7)] and thrombocytopenia [OR = 8.1 (95% CI:1.7-38.8)] were associated with DENV cases at time of presentation. These findings may help clinicians in differentiating CHIKV from DENV infection.Conclusion: CHIKV is an important cause of illness amongst patients presenting with acute febrile illness in Sindh region of Pakistan. Arthralgia and encephalitis at time of presentation among patients with dengue-like illness should prompt suspicion of CHIKV infection, and laboratory confirmation must be sought

Human west nile virus disease outbreak in Pakistan, 2015–2016

Like most of the world, Pakistan has seen an increase in mosquito-transmitted diseases in recent years. The magnitude and distribution of these diseases are poorly understood as Pakistan does not have a nation-wide system for reporting disease. A cross-sectional study to determine which flaviviruses were causing of arboviral disease in Pakistan was instituted. West Nile virus (WNV) is a cause of seasonal fever with neurotropic findings in countries that share borders with Pakistan. Here, we describe the active and persistent circulation of WNV in humans in the southern region of Pakistan. This is the first report of WNV causing neurological disease in human patients in this country. Of 997 enrolled patients presenting with clinical features suggestive of arboviral disease, 105 were positive for WNV IgM antibodies, and 71 of these patients possessed WNV-specific neutralizing antibodies. Cross-reactivity of WNV IgM antibodies with Japanese encephalitis virus(JEV) occurred in 75 of these 105 patients. WNV co-infections with Dengue viruses were not a contributing factor for the severity of disease. Nor did prior exposure to dengue virus contribute to incidence of neurological involvement in WNV-infected patients. Patients with WNV infections were more likely to present with altered mental status, seizures, and reduced Glasgow Coma scores when compared with JEV-infected patients. Human WNV cases and vector numbers exhibited a temporal correlation with climate

Flaviviruses as a cause of undifferentiated fever in Sindh province, Pakistan: A preliminary report

Arboviral diseases are expanding worldwide, yet global surveillance is often limited due to diplomatic and cultural barriers between nations. With human encroachment into new habitats, mosquito-borne viruses are also invading new areas. The actual prevalence of expanding arboviruses is unknown in Pakistan due to inappropriate diagnosis and poor testing for arboviral diseases. The primary objective of this study was to document evidence of flavivirus infections as the cause of undifferentiated fever in Pakistan. Through a cooperative effort between the USA and Pakistan, patient exposure to dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was examined in Sindh Province for the first time in decades. Initial results from the 2015 arbovirus season consisting of a cross-sectional study of 467 patients in 5 sites, DENV NS1 antigen was identified in 63 of the screened subjects, WNV IgM antibodies in 16 patients, and JEV IgM antibodies in 32 patients. In addition, a number of practical findings were made including (1) in silico optimization of RT-PCR primers for flavivirus strains circulating in the Middle East, (2) shipping and storage of RT-PCR master mix and other reagents at ambient temperature, (3) Smart phone applications for the collection of data in areas with limited infrastructure, and (4) fast and reliable shipping for transport of reagents and specimens to and from the Middle East. Furthermore, this work is producing a group of highly trained local scientists and medical professionals disseminating modern scientific methods and more accurate diagnostic procedures to the community

Viral Enrichment Methods Affect the Detection but Not Sequence Variation of West Nile Virus in Equine Brain Tissue

West Nile virus (WNV), a small, positive sense, single stranded RNA virus continues to encroach into new locales with emergence of new viral variants. Neurological disease in the equine can be moderate to severe in the face of low to undetectable virus loads. Physical methods of virus enrichment may increase sensitivity of virus detection and enhance analysis of viral diversity, especially for deep sequencing studies. However, the use of these techniques is limited mainly to non-neural tissues. We investigated the hypothesis that elimination of equine brain RNA enhances viral detection without limiting viral variation. Eight different WNV viral RNA enrichment and host RNA separation methods were evaluated to determine if elimination of host RNA enhanced detection of WNV and increase the repertoire of virus variants for sequencing. Archived brain tissue from 21 different horses was inoculated with WNV, homogenized, before enrichment and separation. The protocols utilized combinations of low-speed centrifugation, syringe filtration, and nuclease treatment. Viral and host RNA were analyzed using real-time PCR targeting the WNV Envelope (E) protein and equine G3PDH to determine relative sensitivity for WNV and host depletion, respectively. To determine the effect of these methods on viral variation, deep sequencing of the E protein was performed. Our results demonstrate that additional separation and enrichment methods resulted in loss of virus in the face of host RNA depletion. DNA sequencing showed no significant difference in total sequence variation between the RNA enrichment protocols. For equine brain infected with WNV, direct RNA extraction followed by host RNA depletion was most suitable. This study highlights the importance of evaluating viral enrichment and separation methods according to tissue type before embarking on studies where quantification of virus and viral variants is essential to the outcome of the study

Microbial Symbionts in Insects Influence Down-Regulation of Defense Genes in Maize

Diabrotica virgifera virgifera larvae are root-feeding insects and significant pests to maize in North America and Europe. Little is known regarding how plants respond to insect attack of roots, thus complicating the selection for plant defense targets. Diabrotica virgifera virgifera is the most successful species in its genus and is the only Diabrotica beetle harboring an almost species-wide Wolbachia infection. Diabrotica virgifera virgifera are infected with Wolbachia and the typical gut flora found in soil-living, phytophagous insects. Diabrotica virgifera virgifera larvae cannot be reared aseptically and thus, it is not possible to observe the response of maize to effects of insect gut flora or other transient microbes. Because Wolbachia are heritable, it is possible to investigate whether Wolbachia infection affects the regulation of maize defenses. To answer if the success of Diabrotica virgifera virgifera is the result of microbial infection, Diabrotica virgifera virgifera were treated with antibiotics to eliminate Wolbachia and a microarray experiment was performed. Direct comparisons made between the response of maize root tissue to the feeding of antibiotic treated and untreated Diabrotica virgifera virgifera show down-regulation of plant defenses in the untreated insects compared to the antibiotic treated and control treatments. Results were confirmed via QRT-PCR. Biological and behavioral assays indicate that microbes have integrated into Diabrotica virgifera virgifera physiology without inducing negative effects and that antibiotic treatment did not affect the behavior or biology of the insect. The expression data and suggest that the pressure of microbes, which are most likely Wolbachia, mediate the down-regulation of many maize defenses via their insect hosts. This is the first report of a potential link between a microbial symbiont of an insect and a silencing effect in the insect host plant. This is also the first expression profile for a plant attacked by a root-feeding insect

Chikungunya in Infants and Children: Is Pathogenesis Increasing?

Chikungunya virus (CHIKV) was first extensively described in children during outbreaks in India and South Asia during the mid-1960s. Prior to the 2005 emergence of CHIKV on Reunion Island, CHIKV infection was usually described as a dengue-like illness with arthralgia in Africa and febrile hemorrhagic disease in Asia. Soon after the 2005 emergence, severe CNS consequences from vertical and perinatal transmission were described and as CHIKV continued to emerge in new areas over the next 10 years, severe manifestation of infection and sequelae were increasingly reported in infants and neonates. The following review describes the global reemergence and the syndromes of Chikungunya fever (CHIKF) in infants and children. The various manifestations of CHIKF are described and connected to the viral lineage that was documented in the area at the time the disease was described. The data show that certain manifestations of CHIKF occur with specific viral lineages and genetic motifs, which suggests that severe manifestations of CHIKF in the very young may be associated with the emergence of new viral lineages

Wolbachia: A screening of potential host organisms [abstract]

Abstract only availableWolbachia are gram-negative bacteria that infect 70% of invertebrates. Previous research in the Davis laboratory, by graduate student Kelli Barr, indicates that WCR-vectored Wolbachia can affect gene expression in Zea mays L. as a consequence of insect feeding. Specifically, Wolbachia turns off host defense genes. Given the ability of WCR-vectored Wolbachia to alter maize gene expression, we wanted to determine whether other pests of maize carry Wolbachia as a first step towards evaluating the extent to which insect-vectored Wolbachia might affect plant gene expression. In addition, several pests of Arabidopsis were also tested for Wolbachia since this plant offers the advantage of shorter generation time. DNA was extracted using a modification of the Qiagen DNEasy kit. The DNA was then amplified via PCR with Wolbachia-specific primers. The PCR products were then electrophoresed on a 0.8% agarose gel with positive and negative controls to determine whether Wolbachia was present. In a second experiment, insects that rapidly overcome chemical control were tested for the presence of Wolbachia and the specific strain of Wolbachia. Since Wolbachia is an obligate parasite in most insects and can induce genetic drive, as well as affect plant gene expression, we hypothesize that the difference in mutation rate and selection pressure on the bacterial compared to the insect genome may explain some insect's ability to easily overcome chemical control. DNA was prepared from these insects and tested for Wolbachia as described above. Wolbachia positive insects were assayed with a standard set of primers to delineate the strain of Wolbachia they carry. Future studies will examine whether Wolbachia vectored by other maize pests can cause similar changes in maize gene expression as those demonstrated with the western corn rootworm

GNSI Decision Brief: Who Will Rebuild Ukraine\u27s Public Health and Disease Research Laboratories?

Amid the devastating war in Ukraine, a parallel battle rages on – that against infectious diseases. The COVID-19 pandemic starkly underlined the vital importance of global health surveillance for emerging and re-emerging human and animal pathogens. This challenge has become even more acute in war-torn regions like Ukraine, where healthcare infrastructure is under siege. For viruses with pandemic potential, such as the influenza virus, worldwide surveillance initiatives are essential in tracking the virus’s movement and spread among both animals and humans. Such monitoring serves dual purposes. Firstly, it guides the formulation of the yearly vaccine, ensuring it remains effective against prevailing strains. Secondly, it alerts livestock farmers in advance, enabling the timely rollout of mitigation measures. Moreover, discerning whether a disease outbreak is of natural origin, or an intentional release becomes critical. To achieve this, the global community must share transparent and timely information through a robust network of laboratories with cutting-edge biotechnological capabilities. The war’s ramifications have severely impacted these efforts. Yet, the stakes are too high to let these facilities fade away. This situation leads to the ultimate question: Who will step up to rebuild Ukraine’s public health and disease research laboratories?https://digitalcommons.usf.edu/gnsi_decision_briefs/1007/thumbnail.jp