Sentinel Enhanced Dengue Surveillance System - Puerto Rico, 2012-2022.

Problem/Condition: Dengue is the most prevalent mosquitoborne viral illness worldwide and is endemic in Puerto Rico. Dengue's clinical spectrum can range from mild, undifferentiated febrile illness to hemorrhagic manifestations, shock, multiorgan failure, and death in severe cases. The disease presentation is nonspecific; therefore, various other illnesses (e.g., arboviral and respiratory pathogens) can cause similar clinical symptoms. Enhanced surveillance is necessary to determine disease prevalence, to characterize the epidemiology of severe disease, and to evaluate diagnostic and treatment practices to improve patient outcomes. The Sentinel Enhanced Dengue Surveillance System (SEDSS) was established to monitor trends of dengue and dengue-like acute febrile illnesses (AFIs), characterize the clinical course of disease, and serve as an early warning system for viral infections with epidemic potential. Reporting Period: May 2012-December 2022. Description of System: SEDSS conducts enhanced surveillance for dengue and other relevant AFIs in Puerto Rico. This report includes aggregated data collected from May 2012 through December 2022. SEDSS was launched in May 2012 with patients with AFIs from five health care facilities enrolled. The facilities included two emergency departments in tertiary acute care hospitals in the San Juan-Caguas-Guaynabo metropolitan area and Ponce, two secondary acute care hospitals in Carolina and Guayama, and one outpatient acute care clinic in Ponce. Patients arriving at any SEDSS site were eligible for enrollment if they reported having fever within the past 7 days. During the Zika epidemic (June 2016-June 2018), patients were eligible for enrollment if they had either rash and conjunctivitis, rash and arthralgia, or fever. Eligibility was expanded in April 2020 to include reported cough or shortness of breath within the past 14 days. Blood, urine, nasopharyngeal, and oropharyngeal specimens were collected at enrollment from all participants who consented. Diagnostic testing for dengue virus (DENV) serotypes 1-4, chikungunya virus, Zika virus, influenza A and B viruses, SARS-CoV-2, and five other respiratory viruses was performed by the CDC laboratory in San Juan. Results: During May 2012-December 2022, a total of 43,608 participants with diagnosed AFI were enrolled in SEDSS; a majority of participants (45.0%) were from Ponce. During the surveillance period, there were 1,432 confirmed or probable cases of dengue, 2,293 confirmed or probable cases of chikungunya, and 1,918 confirmed or probable cases of Zika. The epidemic curves of the three arboviruses indicate dengue is endemic; outbreaks of chikungunya and Zika were sporadic, with case counts peaking in late 2014 and 2016, respectively. The majority of commonly identified respiratory pathogens were influenza A virus (3,756), SARS-CoV-2 (1,586), human adenovirus (1,550), respiratory syncytial virus (1,489), influenza B virus (1,430), and human parainfluenza virus type 1 or 3 (1,401). A total of 5,502 participants had confirmed or probable arbovirus infection, 11,922 had confirmed respiratory virus infection, and 26,503 had AFI without any of the arboviruses or respiratory viruses examined. Interpretation: Dengue is endemic in Puerto Rico; however, incidence rates varied widely during the reporting period, with the last notable outbreak occurring during 2012-2013. DENV-1 was the predominant virus during the surveillance period; sporadic cases of DENV-4 also were reported. Puerto Rico experienced large outbreaks of chikungunya that peaked in 2014 and of Zika that peaked in 2016; few cases of both viruses have been reported since. Influenza A and respiratory syncytial virus seasonality patterns are distinct, with respiratory syncytial virus incidence typically reaching its annual peak a few weeks before influenza A. The emergence of SARS-CoV-2 led to a reduction in the circulation of other acute respiratory viruses. Public Health Action: SEDSS is the only site-based enhanced surveillance system designed to gather information on AFI cases in Puerto Rico. This report illustrates that SEDSS can be adapted to detect dengue, Zika, chikungunya, COVID-19, and influenza outbreaks, along with other seasonal acute respiratory viruses, underscoring the importance of recognizing signs and symptoms of relevant diseases and understanding transmission dynamics among these viruses. This report also describes fluctuations in disease incidence, highlighting the value of active surveillance, testing for a panel of acute respiratory viruses, and the importance of flexible and responsive surveillance systems in addressing evolving public health challenges. Various vector control strategies and vaccines are being considered or implemented in Puerto Rico, and data from ongoing trials and SEDSS might be integrated to better understand epidemiologic factors underlying transmission and risk mitigation approaches. Data from SEDSS might guide sampling strategies and implementation of future trials to prevent arbovirus transmission, particularly during the expansion of SEDSS throughout the island to improve geographic representation.

Reduced spread of influenza and other respiratory viral infections during the COVID-19 pandemic in southern Puerto Rico.

INTRODUCTION: Impacts of COVID-19 mitigation measures on seasonal respiratory viruses is unknown in sub-tropical climates. METHODS: We compared weekly testing and test-positivity of respiratory infections in the 2019-2020 respiratory season to the 2012-2018 seasons in southern Puerto Rico using Wilcoxon signed rank tests. RESULTS: Compared to the average for the 2012-2018 seasons, test-positivity was significantly lower for Influenza A (p<0.001) & B (p<0.001), respiratory syncytial virus (RSV) (p<0.01), respiratory adenovirus (AdV) (p<0.05), and other respiratory viruses (p<0.001) following March 2020 COVID-19 stay at home orders. CONCLUSIONS: Mitigation measures and behavioral social distancing choices may have reduced respiratory viral spread in southern Puerto Rico.

Novel Molecular Signatures of Chikungunya Virus in Puerto Rico.

OBJECTIVE: The chikungunya virus (CHIKV) is an arthropod-borne Alphavirus transmitted to humans, primarily via Aedes mosquitoes. In Puerto Rico, the first locally transmitted infections were reported in May 2014. Although the virus strain in Puerto Rico is related to the Asian/American lineage, many autochthonous cases have emerged recently in the Caribbean region (including Puerto Rico), raising the question of how CHIKV will evolve and adapt in PR. Taking the role of the envelope glycoprotein (E1) in viral evolution and transmission as a given, we analyzed the genetic diversity of the Puerto Rican (PR) E1 gene sequences and the phylogenetic relationships between those sequences and sequences from other parts of the world. MATERIALS AND METHODS: To analyze the overall genetic variation, 772 nucleotide sequences of the E1 gene were obtained from the Virus Pathogen Resource (ViPR). A maximum-likelihood analysis was performed to determine the phylogenetic relationships between the PR sequences and sequences from 48 countries around the world. RESULTS: The analysis of the E1 gene identified variations at 4 nucleotide positions, which included synonymous and nonsynonymous mutations. In addition, 2 nonsynonymous amino acid changes, T207M and S120L, were unique to the PR CHIKV sequences, and T155I was found to be shared by the PR (n = 3) and Colombia (n = 1) strains. CONCLUSION: Our analysis of the E1 gene revealed new molecular signatures in PR CHIKV sequences, 1 of which was also found in Colombia. While studies have shown possible relationships between T98A and A226V with viral adaptation and spread, no other PR sequence contained these vector-adaptive mutations. Thus, constant monitoring of the virus remains an essential factor in the establishment of control strategies to track viral spread.

Cigarette Smoking Modulation of Saliva Microbial Composition and Cytokine Levels.

Tobacco use has been implicated as an immunomodulator in the oral cavity and contributes to the development of oral cancer. In the present study, we investigated the effects of cigarette smoking on bacterial diversity and host responses compared to healthy nonsmoking controls. Saliva samples were collected from eighteen smokers and sixteen nonsmoking individuals by passive drool. The 16S rRNA gene was used to characterize the salivary microbiome by using the Illumina MiSeq platform. Cytokine and chemokine expression analyses were performed to evaluate the host response. Significant differences in cytokine and chemokine expression levels of MDC, IL-10, IL-5, IL-2, IL-4, IL-7, adrenocorticotropic hormone (ACTH), insulin, and leptin were observed between smokers and nonsmokers. Taxonomic analyses revealed differences between the two groups, and some bacterial genera associated with the smokers group had correlations with hormones and cytokines identified as statistically different between smokers and nonsmokers. These factors have been associated with inflammation and carcinogenesis in the oral cavity. The data obtained may aid in the identification of the interactions between the salivary microbiome, host inflammatory responses, and metabolism in smokers.

The Probiotic Mixture VSL#3 Alters the Morphology and Secretion Profile of Both Polarized and Unpolarized Human Macrophages in a Polarization-Dependent Manner.

BACKGROUND: Patients with Inflammatory Bowel Disease (IBD), most commonly Crohn's disease (CD) or ulcerative colitis (UC), suffer from chronic intestinal inflammation of unknown etiology. Increased proinflammatory macrophages (M1) have been documented in tissue from patients with CD. Anti-inflammatory macrophages (M2) may play a role in UC given the preponderance of Th2 cytokines in this variant of IBD. Animal and clinical studies have shown that the probiotic VSL#3 can ameliorate signs and symptoms of IBD. Although animal data suggests a modulatory effect on macrophage phenotype, the effect of VSL#3 on human macrophages remains unknown. OBJECTIVE: To determine the effect of the probiotic VSL#3 on the phenotype of polarized (M1/M2) and unpolarized (MΦ) human macrophages. METHODS: Human monocyte-derived macrophages, generated by culturing monocytes with M-CSF, were left unpolarized or were polarized towards an M1 or an M2 phenotype by culture with LPS and IFN-γ or IL-4, respectively, and were then cultured in the presence or absence of VSL#3 for 3 days. Changes in macrophage morphology were assessed. Cytokine and chemokine levels in supernatants were determined by multiplex assay. RESULTS: VSL#3 decreased the granuloma-like aggregates of M1 macrophages, increased fibroblast-like M2 macrophages, and decreased fibroblast-like MΦ macrophages. VSL#3 increased the secretion of IL-1ß, IL-6, IL-10, and G-CSF by M1, M2, and MΦ macrophages. VSL#3 exposure maintained the proinflammatory phenotype of M1 macrophages, sustaining IL-12 secretion, increasing IL-23 secretion, and decreasing MDC secretion. Both VSL#3-treated M2 and MΦ macrophages secreted higher levels of anti-inflammatory and pro-healing factors such as IL-1Ra, IL-13, EGF, FGF-2, TGF-α, and VEGF, as well as proinflammatory cytokines, including IL-12 and TNF-α. CONCLUSION: Under our experimental conditions VSL#3 induced a mixed proinflammatory and anti-inflammatory phenotype in polarized and unpolarized macrophages. This differential effect could explain why patients with CD do not respond to probiotic therapy as well as patients with UC.

Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory.

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment.

HIV-1 gp120 induces expression of IL-6 through a nuclear factor-kappa B-dependent mechanism: suppression by gp120 specific small interfering RNA.

In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3±2.1 and 11.6±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-κB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKß inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-κB specific siRNA. We also showed that gp120 could increase the phosphorylation of IκBα. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-κB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-κB pathway.

Accelerated evolution of SIV env within the cerebral compartment in the setting of morphine-dependent rapid disease progression.

Human immunodeficiency virus-1 (HIV-1) and simian immunodeficiency virus (SIV) have been shown to compartmentalize within various tissues, including the brain. However, the evolution of viral quasispecies in the setting of drug abuse has not been characterized. The goal of this study was to examine viral evolution in the cerebral compartment of morphine-dependent and control macaques to determine its role in rapid disease progression. To address this issue, we analyzed the envelope (env) gene from proviral DNA in our SIV/SHIV macaque model of morphine dependence and AIDS. Analyses of proviral DNA revealed a direct correlation between total genetic changes and survival time. However, the rate of evolution during disease progression was higher in morphine-dependent and rapid-progressor macaques than was the rate of evolution in the control animals. This study provides additional insight into SIV envelope variation in the CNS of morphine-dependent macaques and genotypes that may have evolved in the brain and contributed to disease progression.

Analysis of the V1V2 region of the SIV envelope in the brains of morphine-dependent and control SIV/SHIV-infected macaques.

Six morphine-dependent and three control macaques were infected with a mixture of SIV/SHIV. Half of the animals in the morphine group developed accelerated disease (rapid progressor) and died within 20 weeks postinfection. The evolution of the envelope gene in the brain of the rapid progressor and morphine-dependent group along with that in the control group was assessed. Six to 10 clones from the brain of each macaque were sequenced and were compared against each other as well as against a challenge virus. Analysis of the sequences revealed that the diversity and divergence of the clones were higher in the control group as compared to the morphine-dependent macaques, although this difference was not statistically significant.

Morphine-mediated deterioration of oxidative stress leads to rapid disease progression in SIV/SHIV-infected macaques.

Oxidative stress is well documented in HIV infection, but the effect of concomitant substance abuse is largely unknown. We studied oxidative stress in our macaque model of morphine abuse and AIDS. In plasma, we found an approximately 50% decrease in catalase activity with morphine dependence that was exacerbated by infection in rapid progressors. Superoxide dismutase was decreased by a similar degree, but only in the presence of both morphine and viral infection. The loss of these antioxidant systems was coincident with significantly increased plasma malondialdehyde upon viral infection that displayed a synergistic increase in conjunction with morphine and rapid disease.

Variable region 4 of SIV envelope correlates with rapid disease progression in morphine-exposed macaques infected with SIV/SHIV.

We analyzed the association between the evolution of the V3-V5 regions of env and disease progression in our SIV/SHIV macaque model of morphine dependence and AIDS. Previous studies revealed two distinct disease patterns--fast progression and normal progression. To determine the effect of the two distinct patterns of disease in the evolution of SIV/17E-Fr envelope, we analyzed env sequences from three morphine-dependent macaques that developed accelerated AIDS and three morphine-dependent macaques that developed AIDS at a slower rate and compared them to control macaques. Morphine-dependent animals exhibited a higher percentage of diversity in both plasma and CSF compartments within V4 when compared to controls. Divergence from the inoculum was significantly greater in the morphine group as compared to controls in CSF but not in plasma. We also found a direct correlation in V4 evolution and rapid disease progression. These results indicate that morphine dependence plays a role in the pathogenesis of SIV/SHIV infection and env evolution.

Secretion of virulence proteins from Campylobacter jejuni is dependent on a functional flagellar export apparatus.

Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.