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Japanese encephalitis virus (JEV) is the emerging and geographically expanding flavivirus and the major causative agent of encephalitis in humans in Asia. There are risks of JEV introduction into the Americas given a large population of amplifying hosts—pigs and wild boars, and insect vectors— Culex mosquitoes. There are emerging concerns about vector-free ways of flavivirus transmission, for example sexual and transplacental Zika virus transmissions, which may change flavivirus epidemiology and expand the geographical range to territories with no insect vectors. It is unknown whether JEV has tropism in the female lower reproductive tract and the potential for sexual transmission in humans. While clinical outcomes of transplacental JEV infection are described in humans and pigs, cellular targets and tissue tropism in the upper reproductive tract are also unknown. Here, we studied JEV infection phenotypes and host transcriptional responses in human reproductive epithelial cells. We found that JEV caused persistent infection and cytopathology in the vaginal epithelium, endometrial epithelium, and trophoblast. Human vaginal epithelial cells infected with JEV had altered transcriptional responses associated with inflammation and disruption of epithelial barrier function. Also, using pigs—the native amplifying host for JEV, we confirmed JEV tropism in the female lower and upper reproductive tracts. We discovered that JEV persists in the vaginal mucosa for at least 28 days and pigs shed the virus in vaginal secretions. We also found JEV persistence in the endometrium and placenta with transplacental and fetal infections. Altogether, we discovered that JEV targets the vaginal epithelium and has the potential for sexual transmission in humans. We also contributed to a better understanding of JEV pathogenesis during transplacental infection. Further studies are needed to better understand the interactions of JEV with reproductive tissues, how persistent infection affects female reproductive functions, and the risks for non-vector transmission.

West Nile virus (WNV) poses a global public health threat. This study demonstrates that the WNV RNA tolerates CpG and UpA dinucleotide enrichment in different genomic regions resulting in attenuation of CpG- and CpG/UpA-enriched variants. Attenuation was zinc finger antiviral protein 1 (ZAP)-dependent, and ZAP knockout (ZAP-KO) cells were used to generate high-titer stocks. Ten enriched variants, with permuted control and wild-type (WT) viruses, were screened in immunocompetent mice upon intraperitoneal injection. In contrast to lethal WNV-WT and permuted viruses, the E-MAX variant, with the RNA region encoding envelope (E) protein enriched both with CpG and UpA, caused no mortality. E-MAX was immunogenic and protective against lethal challenge. Stability of enriched dinucleotides was confirmed upon serial passaging in ZAP-WT and ZAP-KO cells, with only minor (17–21%) reversion at a single site in ZAP-WT condition. E-MAX upregulated interferon (IFN) signaling genes in human cells, suggesting that the combination of CpG/UpA-mediated attenuation, and concurrent activation of IFN responses potentially driven by CpG/UpA enrichment, may contribute to E-MAX immunogenicity. Evaluation using footpad injection in mice showed E-MAX had a promising safety and immunogenicity profile, although brain infection was occasionally detected. Then, we developed the E-MAX+ FR variant by combining CpG/UpA enrichment with two amino acid substitutions in functional domains of the E protein. This strategy eliminated neuroinvasion while maintaining immunogenicity and protection. Altogether, CpG/UpA dinucleotide enrichment in the genomic E region in combination with amino acid substitutions in the E protein yields a promising platform for vaccine development against WNV and potentially other flaviviruses.

Increasing the number of CpG dinucleotides in RNA viral genomes, while preserving the original amino acid composition, leads to impaired infection which does not cause disease. Beneficially, impaired infection evokes antiviral host immune responses providing a cutting-edge vaccine approach. For example, we previously showed that CpG-enriched Zika virus variants cause attenuated infection phenotypes and protect against lethal challenge in mice. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses in vivo , particularly in non-rodent species. Here, we used well-established mouse and porcine models to study infection phenotypes of the CpG-enriched neurotropic and congenital virus—Zika virus, directly in the target tissues—the brain and placenta. Specifically, we used the uttermost challenge and directly injected mice intracerebrally to compare infection phenotypes caused by wild-type and two CpG-recoded Zika variants and model the scenario where vaccine strains breach the blood-brain barrier. Also, we directly injected porcine fetuses to compare in utero infection phenotypes and model the scenario where recoded vaccine strains breach the placental barrier. While overall infection kinetics were comparable between wild-type and recoded virus variants, we found convergent phenotypical differences characterized by reduced pathology in the mouse brain and reduced replication of CpG-enriched variants in fetal lymph nodes. Next, using next-generation sequencing for the whole virus genome, we compared the stability of de novo introduced CpG dinucleotides during prolonged virus infection in the brain and placenta. Most de novo introduced CpG dinucleotides were preserved in sequences of recoded Zika viruses showing the stability of vaccine variants. Altogether, our study emphasized further directions to fine-tune the CpG recoding vaccine approach for better safety and can inform future immunization strategies.

The zinc finger antiviral protein 1 (ZAP) has broad antiviral activity. ZAP is an interferon (IFN)-stimulated gene, which itself may enhance type I IFN antiviral response. In a previous study, Zika virus was identified as ZAP-resistant and not sensitive to ZAP antiviral activity. Here, we found that ZAP was associated with the inhibition of Zika virus in Vero cells, in the absence of a robust type I IFN system because Vero cells are deficient for IFN-alpha and -beta. Also, quantitative RNA-seq data indicated that endogenous ZAP is associated with altered global gene expression both in the steady state and during Zika virus infection. Further studies are warranted to elucidate this IFN-alpha and -beta independent anti-Zika virus activity and involvement of ZAP.

Background Porcine reproductive and respiratory syndrome virus (PRRSV) is divided into a European and North American genotype. East European PRRSV isolates have been found to be of the European genotype, but form different subtypes. In the present study, PRRSV was isolated from a Belarusian farm with reproductive and respiratory failure and designated \"Lena\". Analyses revealed that Lena is a new East European subtype 3 PRRSV isolate. The main purpose of this investigation was to study the pathogenesis and antigenic characteristics of PRRSV (Lena). Results Obvious clinical and virological differences were observed between the animals inoculated with a recent European subtype 1 PRRSV isolate (Belgium A) and animals inoculated with PRRSV (Lena). Three out of six pigs inoculated with PRRSV (Belgium A) had anorexia and low fever at 3, 4 and 5 days post-inoculation (dpi). High fever, anorexia and depression were prominent signs in most pigs inoculated with PRRSV (Lena) between 2 and 28 dpi. Four pigs out of ten died during the experiment. Arcanobacterium pyogenes was isolated from lungs of one animal that died, and Streptococcus suis was isolated from lungs of one animal that was euthanized. The difference in viral titres in sera from PRRSV (Belgium A) and PRRSV (Lena)-infected pigs was statistically significant (p < 0.05) at 7, 10, 14 and 21 dpi. The highest viral titres in sera ranged from 10 4.8 to 10 6.1 TCID 50 /ml for PRRSV (Lena) whereas they ranged from 10 3.1 to 10 4.8 TCID 50 /ml for PRRSV (Belgium A). The replication of PRRSV (Lena) was further studied in depth. Viral titres ranged from 10 2.5 TCID 50 /100 mg to 10 5.6 TCID 50 /100 mg in nasal secretions between 3 and 14 dpi and from 10 2.8 TCID 50 /100 mg to 10 4.6 TCID 50 /100 mg in tonsillar scrapings between 3 and 21 dpi. High viral titres were detected in lungs (10 2.3 -10 7.7 TCID 50 /g tissue), tonsils (10 2.0 -10 6.2 TCID 50 /g tissue) and inguinal lymph nodes (10 2.2 -10 6.6 TCID 50 /g tissue) until 35, 28 and 35 dpi, respectively. To examine the antigenic heterogeneity between the East European subtype 3 isolate Lena, the European subtype 1 strain Lelystad and the North American strain US5, sets of monospecific polyclonal antisera were tested in immunoperoxidase monolayer assays (IPMAs) with homologous and heterologous viral antigens. Heterologous antibody titres were significantly lower than homologous titres (p = 0.01-0.03) for antisera against PRRSV (Lena) at all sampling time points. For antisera against PRRSV (Lelystad) and PRRSV (US5), heterologous antibody titres were significantly lower than homologous titres at 14 and 21 dpi (p = 0.01-0.03) and at 10 and 14 dpi (p = 0.04), respectively. Conclusions Lena is a highly pathogenic East European subtype 3 PRRSV, which differs from European subtype 1 Lelystad and North American US5 strains at both the genetic and antigenic level.

We studied whether cytosine phosphate–guanine (CpG) recoding in a viral genome may provide oncolytic candidates with reduced infection kinetics in nonmalignant brain cells, but with high virulence in glioblastoma stem cells (GSCs). As a model, we used well-characterized CpG-recoded Zika virus vaccine candidates that previously showed genetic stability and safety in animal models. In vitro, one of the CpG-recoded Zika virus variants had reduced infection kinetics in nonmalignant brain cells but high infectivity and oncolytic activity in GSCs as represented by reduced cell proliferation. The recoded virus also efficiently replicated in GSC-derived tumors in ovo with a significant reduction of tumor growth. We also showed that some GSCs may be resistant to Zika virus oncolytic activity, emphasizing the need for personalized oncolytic therapy or a strategy to overcome resistance in GSCs. Collectively, we demonstrated the potential of the CpG recoding approach for oncolytic virus development that encourages further research towards a better understanding of host–tumor–CpG-recoded virus interactions.

Studies in mice showed that African Zika virus (ZIKV) strains cause more damage in embryos. These studies, however, were limited to the mouse-adapted African MR766 strain or infection at early gestation. Here, we compared infection of Asian and African strains in the fetal pig model at midgestation. Both strains caused fetal infection. ZIKV was detected in placenta, amniotic membrane, amniotic fluid, fetal blood, and brain. The African strain produced more vigorous in utero infection as represented by more efficient virus transmission between siblings, and higher viral loads in fetal organs and membranes. Infection with both strains was associated with reduced fetal brain weight and increased number of placental CD163-positive cells, as well as elevated in utero interferon alpha and cortisol levels. This is the first large animal model study which demonstrated that African strain of ZIKV, with no passage history in experimental animals, can cause persistent infection in fetuses and fetal membranes at midgestation. Our studies also suggest that similar to Asian strains, ZIKV of African lineage might cause silent pathology which is difficult to identify in deceptively healthy fetuses. The findings emphasize the need for further studies to highlight the impact of ZIKV heterogeneity on infection outcomes during pregnancy.

Recoding strategies have emerged as a promising approach for developing safer and more effective vaccines by altering the genetic structure of microorganisms, such as viruses, without changing their proteins. This method enhances vaccine safety and efficacy while minimizing the risk of reversion to virulence. Recoding enhances the frequency of CpG dinucleotides, which in turn activates immune responses and ensures a strong attenuation of the pathogens. Recent advancements highlight synonymous recoding’s potential, offering improved genetic stability and immunogenicity compared to traditional methods. Live vaccines attenuated using classical methods pose a risk of reversion to virulence and can be time-consuming to produce. Synonymous recoding, involving numerous codon alterations, boosts safety and vaccine stability. One challenge is balancing attenuation with yield; however, innovations like Zinc-finger antiviral protein (ZAP) knockout cell lines can enhance vaccine production. Beyond viral vaccines, recoding can apply to bacterial vaccines, as exemplified by modified Escherichia coli and Streptococcus pneumoniae strains, which show reduced virulence. Despite promising results, challenges like ensuring genetic stability, high yield, and regulatory approval remain. Briefly, ongoing research aims to harness these innovations for comprehensive improvements in vaccine design and deployment. In this commentary, we sought to further engage the community’s interest in this elegant approach by briefly highlighting its main advantages, disadvantages, and future prospects.

Congenital virus infections, for example cytomegalovirus and rubella virus infections, commonly affect the central nervous and hematological systems in fetuses and offspring. However, interactions between emerging congenital Zika virus and hematological system-bone marrow and blood-in fetuses and offspring are mainly unknown. Our overall goal was to determine whether silent in utero Zika virus infection can cause functional and molecular footprints in the bone marrow and blood of fetuses and offspring. We specifically focused on silent fetal infection because delayed health complications in initially asymptomatic offspring were previously demonstrated in animal and human studies. Using a well-established porcine model for Zika virus infection and a set of cellular and molecular experimental tools, we showed that silent in utero infection causes multi-organ inflammation in fetuses and local inflammation in the fetal bone marrow. In utero infection also caused footprints in the offspring bone marrow and PBMCs. These findings should be considered in a broader clinical context because of growing concerns about health sequelae in cohorts of children affected with congenital Zika virus infection in the Americas. Understanding virus-induced molecular mechanisms of immune activation and inflammation in fetuses may provide targets for early in utero interventions. Also, identifying early biomarkers of in utero-acquired immunopathology in offspring may help to alleviate long-term sequelae.

Experimental increase of CpG dinucleotides in an RNA virus genome impairs infection providing a promising approach for vaccine development. While CpG recoding is an emerging and promising vaccine approach, little is known about infection phenotypes caused by recoded viruses . For example, infection phenotypes, immunogenicity, and protective efficacy induced by CpG-recoded viruses in different age groups were not studied yet. This is important, because attenuation of infection phenotypes caused by recoded viruses may depend on the population-based expression of cellular components targeting viral CpG dinucleotides. In the present study, we generated several Zika virus (ZIKV) variants with the increasing CpG content and compared infection in neonatal and adult mice. Increasing the CpG content caused host-age-dependent attenuation of infection with considerable attenuation in neonates and high attenuation in adults. Expression of the zinc-finger antiviral protein (ZAP)-the host protein targeting viral CpG dinucleotides-was also age-dependent. Similar to the wild-type virus, ZIKV variants with the increased CpG content evoked robust cellular and humoral immune responses and protection against lethal challenge. Collectively, the host age should be accounted for in future studies on mechanisms targeting viral CpG dinucleotides, development of safe dinucleotide recoding strategies, and applications of CpG-recoded vaccines.