After the unexpected arrival of West Nile virus (WNV) in the United States in 1999, the mosquito-borne virus quickly spread throughout North America. Over the past 20 years, WNV has become endemic, with sporadic epizootics. Concerns about the economic impact of infection in horses lead to the licensure of an equine vaccine as early as 2005, but few advances regarding human vaccines or treatments have since been made. There is a high level of virus transmission in hot/humid, subtropical climates, and high morbidity that may disproportionately affect vulnerable populations including the homeless, elderly, and those with underlying health conditions. Although WNV continues to cause significant morbidity and mortality at great cost, funding and research have declined in recent years. These factors, combined with neglect by policy makers and amenability of control measures, indicate that WNV has become a neglected tropical disease.

We conducted preclinical studies in mice using a yeast-produced SARS-CoV-2 RBD subunit vaccine candidate formulated with aluminum hydroxide (alum) and CpG deoxynucleotides. This formulation is equivalent to the Corbevax^TM vaccine that recently received emergency use authorization by the Drugs Controller General of India. We compared the immune response of mice vaccinated with RBD/alum to mice vaccinated with RBD/alum + CpG. We also evaluated mice immunized with RBD/alum + CpG and boosted with RBD/alum. Mice were immunized twice intramuscularly at a 21-day interval. Compared to two doses of the /alum formulation, the RBD/alum + CpG vaccine induced a stronger and more balanced Th1/Th2 cellular immune response, with high levels of neutralizing antibodies against the original Wuhan isolate of SARS-CoV-2 as well as the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 and (Delta) variants. Neutralizing antibody titers against the B.1.1.529 (BA.1, Omicron) variant exceeded those in human convalescent plasma after Wuhan infection but were lower than against the other variants. Interestingly, the second dose did not benefit from the addition of CpG, possibly allowing dose-sparing of the adjuvant in the future. The data reported here reinforces that the RBD/alum + CpG vaccine formulation is suitable for inducing broadly neutralizing antibodies against SARS-CoV-2, including variants of concern.

West Nile virus (WNV) is an important emerging flavivirus in North America. Experimental studies in animals infer the development of persistent infection in the kidneys. In humans, recent studies suggest the possibility of persistent renal infection and chronic kidney disease. Considering the discrepancies between published studies on viral RNA detection in urine of convalescing WNV-positive patients, we explored the use of electron microscopy (EM) with anti-WNV E protein antibody immunogold labeling to detect virus in the urine sediment from a subset of study participants in the Houston WNV cohort. In 42% of evaluated study participants had visible sediment present in urine after centrifugation; viral particles consistent with the size and morphology of WNV were successfully detected using EM in the urine sediment up to 9 years postinfection. The anti-WNV immunogold labeling bound to virus envelope in the sediment allowed for enhanced detection when compared with PCR and provide a new technique for understanding kidney disease in WNV patients. These results provide further evidence of persistent infection in at least a subset of individuals infected with WNV. These findings present a novel tool to diagnose persistent WNV infection and its possible link with progressive renal pathology.

Aim: This study investigated the stability of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on 16 common environmental surface materials. Background: SARS-CoV-2 is the causative agent of severe coronavirus disease, a significant public health concern that quickly led to a pandemic. Contamination of environmental surface materials is of concern, with previous studies identifying long-term detection of infectious particles on surfaces. These contaminated surfaces create an increased risk for contact transmission. Methods: Surface materials were inoculated with 10,000 plaque forming units and samples were collected 4, 8, 12, 24, 30, 48, and 168 hours post infection (hpi). Viral titers were determined for each sample and time point using plaque assays. Nonparametric modeling utilized the Turnbull algorithm for interval-censored data. Maximum likelihood estimates for the survival curve were calculated. Parametric proportional hazards regression models for interval censored data were used to explore survival time across the surface materials. Results: There was a sharp decline in recoverable virus after 4 hpi for all tested surfaces. By 12 hpi, infectious SARS-CoV-2 was recoverable from only four surfaces; and by 30 hr, the virus was recoverable from only one surface. There were differences in survival curves based on the materials although some groups of materials are similar, both statistically and practically. Conclusions: While very low amounts of infectious SARS-CoV-2 are recoverable over time, there remains a risk of viral transmission by surface contamination in indoor environments. Individuals and institutions must follow appropriate procedures to decontaminate indoor environment and increase diligence for hand hygiene and personal protective equipment.

West Nile virus (WNV), a mosquito-borne arbovirus, remains a major global health concern. In this study, we optimized PCR methods then assessed serially-collected whole blood (WB), urine (UR), saliva, and semen specimens from a large cohort of WNV-positive participants to evaluate the natural history of infection and persistent shedding of WNV RNA. Viral RNA extraction protocols for frozen WB and UR specimens were optimized and validated through spiking experiments to maximize recovery of viral RNA from archived specimens and to assess the degradation of WNV RNA in stored UR specimens. The resultant procedures were used in conjunction with PCR detection to identify WNV-positive specimens and to quantify their viral loads. A total of 59 of 352 WB, 10 of 38 UR, and 2 of 34 saliva specimens tested positive for WNV RNA. Although a single semen specimen was positive 22 days post onset, we could not definitively confirm the presence of WNV RNA in the remaining specimens. WNV RNA-positive UR specimens exhibited profound loss of viral RNA during storage, highlighting the need for optimal preservation pre-storage. This study provides optimized methods for WNV RNA detection among different fluid types and offers alternative options for diagnostic testing during the acute stages of WNV.

Current diagnostic protocols of acute Zika virus (ZIKV) infection focus on detection of viral RNA in serum or urine using reverse transcription quantitative polymerase chain reaction (RT-qPCR); however, detecting infection can be a challenge, given that 80% of people with acute ZIKV infection are asymptomatic, and the window to detect viremia in serum is short. The ability to extend that window is needed to detect ZIKV at later time points after infection, particularly in high-risk individuals such as pregnant women. We evaluated RNA extraction methods to optimize detection of ZIKV in various body fluids using RT-qPCR as a means of improving the analytical sensitivity of detection. We optimized methods for ZIKV RNA recovery from a number of body fluids by spiking with three varying concentrations of virus, then comparing recovery with that of spiked buffer control. RNA extraction protocols were adjusted as necessary for maximum RNA recovery. Adjustment of the elution step was essential for improved ZIKV RNA recovery from whole blood, saliva, vaginal secretions, and breast milk. Optimal recovery from urine samples required the addition of Urine Conditioning Buffer, and the use of RLT Plus buffer and RNeasy Mini Spin Columns was necessary for RNA extractions from semen samples. Optimized QIAamp MinElute Virus Spin Kit (QIAGEN, Valencia, CA) protocol followed by the singleplex ZIKV RT-qPCR assay provided a reliable method for detection of ZIKV RNA in a variety of biological samples. Improved diagnostics are crucial for timely detection and diagnosis, particularly during pregnancy when the consequences of ZIKV infection can greatly impact the developing fetus.