Negevirus Piura Suppresses Zika Virus Replication in Mosquito Cells.

We investigated the interaction between the insect-specific virus, Piura virus (PIUV), and the arbovirus Zika virus (ZIKV) in Aedes albopictus cells. We performed coinfection experiments in C6/36 cells. Piura virus (Cor 33 strain, Colombia) and ZIKV (PRVABC58 strain, Puerto Rico) were co-inoculated into C6/36 cells using two multiplicity of infection (MOI) combinations: 0.1 for both viruses and 1.0 for ZIKV, 0.1 for PIUV. Wells were infected in triplicate with either PIUV and ZIKV coinfection, ZIKV-only, or PIUV-only. Mock infected cells served as control wells. The cell suspension was collected daily 7 days post-infection. Zika virus load was titrated by TCID50 on Vero 76 cells. The ZIKV-only infection and PIUV and ZIKV coinfection experiments were also quantified by RT-qPCR. We also investigated whether ZIKV interfered in the PIUV replication. PIUV suppressed the replication of ZIKV, resulting in a 10,000-fold reduction in ZIKV titers within 3 days post-infection. PIUV viral loads were not reduced in the presence of ZIKV. We conclude that, when concurrently infected, PIUV suppresses ZIKV in C6/36 cells while ZIKV does not interfere in PIUV replication.

Leptospira transcriptome sequencing using long-read technology reveals unannotated transcripts and potential polyadenylation of RNA molecules.

IMPORTANCE: Leptospirosis, caused by the spirochete bacteria Leptospira, is a zoonotic disease of humans and animals, accounting for over 1 million annual human cases and over 60,000 deaths. We have characterized operon transcriptional units, identified novel RNA coding regions, and reported evidence of potential posttranscriptional polyadenylation in the Leptospira transcriptomes for the first time using Oxford Nanopore Technology RNA sequencing protocols. The newly identified RNA coding regions and operon transcriptional units were detected only in the pathogenic Leptospira transcriptomes, suggesting their significance in virulence-related functions. This article integrates bioinformatics, infectious diseases, microbiology, molecular biology, veterinary sciences, and public health. Given the current knowledge gap in the regulation of leptospiral pathogenicity, our findings offer valuable insights to researchers studying leptospiral pathogenicity and provide both a basis and a tool for researchers focusing on prokaryotic molecular studies for the understanding of RNA compositions and prokaryotic polyadenylation for their organisms of interest.

Everglades virus evolution: Genome sequence analysis of the envelope 1 protein reveals recent mutation and divergence in South Florida wetlands.

Everglades virus (EVEV) is a subtype (II) of Venezuelan equine encephalitis virus (VEEV), endemic in southern Florida, USA. EVEV has caused clinical encephalitis in humans, and antibodies have been found in a variety of wild and domesticated mammals. Over 29,000 Culex cedecei females, the main vector of EVEV, were collected in 2017 from Big Cypress and Fakahatchee Strand Preserves in Florida and pool-screened for the presence of EVEV using reverse transcription real-time polymerase chain reaction. The entire 1 E1 protein gene was successfully sequenced from fifteen positive pools. Phylogenetic analysis showed that isolates clustered, based on the location of sampling, into two monophyletic clades that diverged in 2009. Structural analyses revealed two mutations of interest, A116V and H441R, which were shared among all isolates obtained after its first isolation of EVEV in 1963, possibly reflecting adaptation to a new host. Alterations of the Everglades ecosystem may have contributed to the evolution of EVEV and its geographic compartmentalization. This is the first report that shows in detail the evolution of EVEV in South Florida. This zoonotic pathogen warrants inclusion into routine surveillance given the high natural infection rate in the vectors. Invasive species, increasing urbanization, the Everglades restoration, and modifications to the ecosystem due to climate change and habitat fragmentation in South Florida may increase rates of EVEV spillover to the human population.

Leptospira enrichment culture followed by ONT metagenomic sequencing allows better detection of Leptospira presence and diversity in water and soil samples.

BACKGROUND: Leptospirosis, a life-threatening disease in humans and animals, is one of the most widespread global zoonosis. Contaminated soil and water are the major transmission sources in humans and animals. Clusters of disease outbreaks are common during rainy seasons. METHODOLOGY/PRINCIPAL FINDINGS: In this study, to detect the presence of Leptospira, we applied PCR, direct metagenomic sequencing, and enrichment culture followed by PCR and metagenomic sequencing on water and soil samples. Direct sequencing and enrichment cultures followed by PCR or sequencing effectively detected pathogenic and nonpathogenic Leptospira compared to direct PCR and 16S amplification-based metagenomic sequencing in soil or water samples. Among multiple culture media evaluated, Ellinghausen-McCullough-Johnson-Harris (EMJH) media containing antimicrobial agents was superior in recovering and detecting Leptospira from the environmental samples. Our results show that enrichment culture followed by PCR can be used to confirm the presence of pathogenic Leptospira in environmental samples. Additionally, metagenomic sequencing on enrichment cultures effectively detects the abundance and diversity of Leptospira spp. from environmental samples. CONCLUSIONS/SIGNIFICANCE: The selection of methodology is critical when testing environmental samples for the presence of Leptospira. Selective enrichment culture improves Leptospira detection efficacy by PCR or metagenomic sequencing and can be used successfully to understand the presence and diversity of pathogenic Leptospira during environmental surveillance.

Galleria mellonella infection model to evaluate pathogenic and nonpathogenic Leptospira strains.

The Galleria mellonella larvae infection model is emerging as a valuable tool for studying various characteristics of infectious agents and host-pathogen interaction. This system has been widely recognized as a high throughput, ethical, and cost-effective invertebrate infection model to study the virulence and pathogenesis of various bacterial pathogens. In this study, we compared the effect of Leptospira infection in G. mellonella larvae infected with Leptospira interrogans serovar Copenhageni (pathogenic) or Leptospira biflexa serovar Patoc (saprophytic) strains. We observed significant pathologic changes such as decreased activity, complete melanization, and lower survival rate in the G. mellonella larvae infected with a pathogenic strain L. interrogans serovar Copenhageni compared to those infected with a nonpathogenic strain L. biflexa serovar Patoc. Our study demonstrates the feasibility and the potential of using G. mellonella larvae as an alternative model to study virulence mechanisms and pathogenesis of Leptospira strains. Once optimized, the G. mellonella infection model can be a potential substitute for hamsters to explore various host and pathogen-related mechanistic events in Leptospira infection.

Feasibility of using tissue autolysis to estimate the postmortem interval in horses.

Estimation of the postmortem interval (PMI) is a poorly studied field in veterinary pathology. The development of field-applicable methods is needed given that animal cruelty investigations are increasing continually. We evaluated various histologic criteria in equine brain, liver, and muscle tissue to aid the estimation of PMI in horses, which is central to forensic investigations of suspicious death. After death, autolysis proceeds predictably, depending on environmental conditions. Currently, no field-applied methods exist that accurately estimate the PMI using histology in animals or humans through quantification of autolysis. Brain, liver, and skeletal muscle from 12 freshly euthanized horses were held at 22°C and 8°C for 72 h. Tissues were sampled at T0h, T1h, T2h, T4h, T6h, T12h, T24h, T36h, T48h, T60h, and T72h. For each tissue, we quantified 5 to 7 criteria associated with autolysis, based on the percentage of microscopic field involved. Each criterion was modeled, with temperature and time as independent variables. Changes were most predictable in liver and muscle over the first 72 h postmortem. The criteria for autolysis that were present most extensively at both temperatures were hepatocyte individualization and the separation of bile duct epithelium from the basement membrane. The changes that were present next most extensively were disruption of myofiber continuity, hypereosinophilia, and loss of striation. Brain changes were highly variable. The high statistical correlation between the parameter "autolysis" and the variables "time/temperature", indicates that autolysis is progressive and predictable. Further investigation of these criteria is needed to establish histologic algorithms for PMI.

Chikungunya outbreak in Karachi Pakistan 2016-2017: An analysis of viral isolates.

In December 2016 physicians in Karachi, Pakistan,witnessed an increase in patients presenting with febrile illness and severe polyarthralgia. Subsequently, chikungunya virus (CHIKV)) was isolated from three patients. This virus was sequenced and compared with other isolates of CHIKV obtained in India and Pakistan during recent outbreaks. Phylogenetic analysis indicated that the Karachi isolates were most similar to the East Central South African CHIKV lineage and showed sequence homology to isolates obtained in other parts of Pakistan and India. More importantly, two of the CHIKV isolates had a nucleotide substitution in the E1 gene corresponding to an amino acid change at chain F portion of the E1 protein.

The Clinical Features of Co-circulating Dengue Viruses and the Absence of Dengue Hemorrhagic Fever in Pakistan.

Dengue virus (DENV) is the most common and widespread arboviral infection worldwide. Though all four DENV serotypes cocirculate in nature, the clinicopathological framework of these serotypes is undefined in Pakistan. A cross-sectional, observational study was performed to document the circulation of various arboviruses in the Sindh region of Pakistan. Here we describe a population of patients diagnosed with DENV spanning a 2-year period. This study used an orthogonal system of NS1 antigen ELISA followed by RT-PCR for DENV detection and subtyping. A total of 168 NS1 positive patients were evaluated of which 91 patients were serotyped via RT-PCR. There was no significant difference between sex or age for infection risk and peak transmission occurred during the Autumn months. DENV2 was the most common serotype followed by DENV1 then DENV3, then DENV4. The data show that DENV1 patients were more likely to have abnormal liver function tests; DENV2 infected patients were more likely to exhibit arthralgia and neurological symptoms; DENV3 patients were more likely to complain of burning micturition and have elevated lymphocyte counts and low hematocrit; and DENV4 patients were more likely to report headaches and rash. Notably, no dengue hemorrhagic fever or other manifestations of severe dengue fever were present in patients with primary or secondary infections. We were able to identify significantly more NS1 antigen positive patients than RT-PCR. This study demonstrates that all four DENV serotypes are co-circulating and co-infecting in Pakistan.

Complete Genome Sequence of a Virulent Leptospira interrogans Serovar Copenhageni Strain, Assembled with a Combination of Nanopore and Illumina Reads.

Here, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage.

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.

Strain-Dependent Activity of Zika Virus and Exposure History in Serological Diagnostics.

Zika virus (ZIKV) circulates as two separate lineages, with significant genetic variability between strains. Strain-dependent activity has been reported for dengue virus, herpes simplex virus and influenza. Strain-dependent activity of subject specimens to a virus could be an impediment to serological diagnosis and vaccine development. In order to determine whether ZIKV exhibits strain-dependent activity when exposed to antibodies, we measured the neutralizing properties of polyclonal serum and three monoclonal antibodies (ZKA185, 753(3)C10, and 4G2) against three strains of ZIKV (MR-766, PRVABC59, and R103454). Here, MR-766 was inhibited almost 60% less by ZKA185 than PRVABC59 and R103454 (p = 0.008). ZKA185 enhanced dengue 4 infection up to 50% (p = 0.0058). PRVABC59 was not inhibited by mAb 753(3)C10 while MR-766 and R103453 were inhibited up to 90% (p = 0.04 and 0.036, respectively). Patient serum, regardless of exposure history, neutralized MR-766 ~30%-40% better than PRVABC56 or R103454 (p = 0.005-0.00007). The most troubling finding was the significant neutralization of MR-766 by patients with no ZIKV exposure. We also evaluated ZIKV antibody cross reactivity with various flaviviruses and found that more patients developed cross-reactive antibodies to Japanese encephalitis virus than the dengue viruses. The data here show that serological diagnosis of ZIKV is complicated and that qualitative neutralization assays cannot discriminate between flaviviruses.

Experimental Infection of Pregnant Female Sheep with Zika Virus During Early Gestation.

Zika virus (ZIKV) is a vertically and sexually transmissible virus resulting in severe congenital malformation. The goal of this study was to develop an ovine model of ZIKV infection. Between 28-35 days gestation (DG), four pregnant animals were infected with two doses of 6 × 106 PFU of ZIKV; four control animals received PBS. Animals were evaluated for 45 days (D) post-infection (PI) and necropsies were performed. Viral RNA was detected in infected ewe peripheral blood mononuclear cells (PBMC) during the first week PI; however, all fluids and tissues were negative upon culture. Anti-ZIKV IgM (1:400) and neutralizing antibodies were detected in all infected animals. Clinical disease, virus, or ZIKV antibodies were not detected in control ewes. After two weeks PI, fetal loss occurred in two infected animals, and at necropsy, three infected animals had placental petechiation and ecchymosis and one had hydramnion. Fetal morphometrics revealed smaller cranial circumference to crown-rump length ratios (p < 0.001) and relative brain weights (p = 0.038) in fetuses of infected animals compared with control fetuses. Immunophenotyping indicated an increase in B cells (p = 0.012) in infected sheep. Additionally, in vitro experiments using both adult and fetal cell lines demonstrated that ovine cells are highly permissive to ZIKV infection. In conclusion, ZIKV infection of pregnant sheep results in a change in fetal growth and gestational outcomes.

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.

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.

Histologic characterization of eosinophilic encephalitis in horses in Florida.

Eosinophils within the central nervous system are abnormal and are usually associated with fungal or parasitic infections in horses. Causative agents include Halicephalobus gingivalis, Sarcocystis neurona, and Neospora hughesi. Confirmation of these organisms via specific testing is typically not performed, and final diagnoses are often presumptive. With molecular technology, many of these organisms can now be confirmed. This is important for emerging and zoonotic pathogens, including Angiostrongylus cantonensis, an emerging parasite of interest in the southeastern United States. We retrospectively analyzed eosinophilic encephalitides in Floridian horses for H. gingivalis, S. neurona, and A. cantonensis, applied descriptors to equine eosinophilic encephalitides, and determined if a relationship existed between these descriptions and specific etiologic agents. In a database search for horses with eosinophilic and other encephalitides submitted to the University of Florida, College of Veterinary Medicine, Anatomic Pathology Service, we identified 27 horses with encephalitis, and performed DNA isolation and rtPCR on formalin-fixed, paraffin-embedded blocks from these cases. Real-time PCR identified 6 horses positive for S. neurona and 4 horses positive for H. gingivalis; all horses were negative for A. cantonensis. All 25 control horses were negative for H. gingivalis, S. neurona, and A. cantonensis. Pattern analysis and eosinophil enumeration were not useful in differentiating among causes of eosinophilic encephalitides in horses in our study.

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.

Corrigendum: Human West Nile Virus Disease Outbreak in Pakistan, 2015-2016.

[This corrects the article DOI: 10.3389/fpubh.2018.00020.].

Working with Zika and Usutu Viruses In Vitro.

Usutu (USUV) and Zika (ZIKV) viruses are emerging arboviruses of significant medical and veterinary importance. These viruses have not been studied as well as other medically important arboviruses such as West Nile (WNV), dengue (DENV), or chikungunya (CHIKV) viruses. As such, information regarding the behavior of ZIKV and USUV viruses in the laboratory is dated. Usutu virus re-emerged in Austria in 2001 and has since spread throughout the European and Asian continents causing significant mortality among birds. Zika virus has recently appeared in the Western Hemisphere and has exhibited high rates of birth defects and sexual transmission. Information about the characteristics of USUV and ZIKV viruses are needed to better understand the transmission, dispersal, and adaptation of these viruses in new environments. Since their initial characterization in the middle of last century, technologies and reagents have been developed that could enhance our abilities to study these pathogens. Currently, standard laboratory methods for these viruses are limited to 2-3 cell lines and many assays take several days to generate meaningful data. The goal of this study was to characterize these viruses in cells from multiple diverse species. Cell lines from 17 species were permissive to both ZIKV and USUV. These viruses were able to replicate to significant titers in most of the cell lines tested. Moreover, cytopathic effects were observed in 8 of the cell lines tested. These data indicate that a variety of cell lines can be used to study ZIKV and USUV infection and may provide an updated foundation for the study of host-pathogen interactions, model development, and the development of therapeutics.

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.