Since its initial isolation in Uganda in 1937 through the present, West Nile virus (WNV) has become an important cause of human and animal disease worldwide. WNV, an enveloped virus of the genusFlavivirus, is naturally maintained in an enzootic cycle between birds and mosquitoes, with occasional epizootic spillover causing disease in humans and horses. The mosquito vectors for WNV are widely distributed worldwide, and the known geographic range of WNV transmission and disease has continued to increase over the past 77 years. While most human infections with WNV are asymptomatic, severe neurological disease may develop resulting in long-term sequelae or death. Surveillance and preventive measures are an ongoing need to reduce the public health impact of WNV in areas with the potential for transmission.

Zika virus (ZIKV) emerged in the Americas in 2015, presenting unique challenges to public health. Unlike other arboviruses of the Flaviviridae family, it is transmissible by sexual contact, which facilitates the spread of the virus into new geographic areas. Additionally, ZIKV can be transmitted from mother to fetus, causing microcephaly and other severe developmental abnormalities. Reliable and easy-to-work-with clones of ZIKV expressing heterologous genes will significantly facilitate studies aimed at understanding the virus pathogenesis and tissue tropism. Here, we developed and characterized two novel approaches for expression of heterologous genes of interest in the context of full-length ZIKV genome and compared them to two previously published strategies for ZIKV-mediated gene expression. We demonstrated that among the four tested viruses expressing nLuc gene, the virus constructed using a newly developed approach of partial capsid gene duplication (PCGD) attained the highest titer in Vero cells and resulted in the highest level of nLuc expression. Suitability of the PCGD approach for expression of different genes of interest was validated by replacing nLuc sequence with that of eGFP gene. The generated constructs were further characterized in cell culture. Potential applications of ZIKV clones stably expressing heterologous genes include development of detection assays, antivirals, therapeutics, live imaging systems, and vaccines.

Zika virus (ZIKV) caused a public health threat in the United States in 2016, leading to rapid development and implementation of blood screening assays for ZIKV RNA. Several ZIKV sequences from clinical cases have been reported, but none from asymptomatic/pre-symptomatic infections. We isolated and sequenced ZIKV from asymptomatic/pre-symptomatic blood donor (ABD-ZIKV) samples and compared with reported clinical sequences. Twelve ABD-ZIKV isolates were produced from 67 cultivated samples, and isolates were genetically similar among themselves. Most isolates shared mutations with the clinical isolate PRVABC59 2015, whereas two ABD-ZIKV isolates shared specific mutations with U.S. clinical isolates from 2016. The ABD-ZIKV strains clustered into two distinct subclades: one comprised mostly ABD-ZIKV from Puerto Rico, and another one comprised ABD-ZIKV from Florida and QTX-02 isolate (Puerto Rico). In this study, we showed the circulation of two slightly distinct virus strains among Puerto Rico blood donors, one of which was also reported in Florida.

Blood transfusion is an effective therapeutic approach for several hematological conditions including sickle cell disease (SCD), thalassaemia, myelodysplastic syndrome (MDS), and autoimmune hemolytic anemia. It is also often indicated for transplantation and for patients receiving medical treatments for cancer. However, transfusion treatment can lead to the red blood cell (RBC) alloimmunization when an incompatible antigen is inadvertently present in the transfused blood. Alloantibodies can cause RBC destruction and many other complications defeating the purpose of the treatment. The risk of development of multiple alloantibodies increases with the frequency of transfusions in transfusion-dependent patients and can be mitigated by transfusing blood type negative for multiple antigens to prevent hemolysis. This chapter discusses the transfusion’s risk of RBC alloimmunization as an adverse event; consequences of alloimmunization in patients’ care; approaches to prevent and/or mitigate alloimmunization and enhance transfusion efficacy; application of RBC genotyping to supplement serology for preventing alloimmunization. The currently available techniques for RBC genotyping and the importance of reference reagents for determining the genotyping accuracy will also be discussed.