H5N6 avian influenza viruses (AIVs) can cause severe pneumonia and death in humans. However, the molecular determinants of H5N6 influenza virus mammalian adaption are still unclear. Three amino acid substitutions (HA A150V, PA A343T, PB2 E627K) are observed in H5N6 virus A/duck/Zhejiang/6D2/2013 (6D2) in lung-to-lung passage in mice. These substitutions are crucial to the pathogenicity of mouse-adapted virus. In this study, we investigated the contribution of each amino acid substitution in the virus by reverse genetics. The results demonstrate that HA A150V greatly altered the receptor binding preference of 6D2. Virus bearing this substitution acquired increased mortality than mice infected with wild-type 6D2. The PA A343T substitution mildly enhanced viral polymerase activity but the reduced survival rate in mice indicates this substitution may change the immunoreaction of the host. The well-known PB2 E627K substitution increased eight folds the relative polymerase activity compared to PA A343T and resulted in 100% death rate in mice. In addition, we show that PA A343T dramatically exacerbates the effect of PB2 E627K on viral polymerase activity; when combined, these two substitutions work synergistically. However, HA A150V and PA A343T seemed to attenuate PB2 E627K in vivo, which implies the difference between mixed viral populations under natural condition and single population under experiment, specialization and cooperation in quasispecies is important in the process of adaption. This study suggests that HA A150V, PA A343T, and PB2 E627K are crucial in the adaption and increased pathogenicity of H5N6 in mammalian hosts.

We assess long-term changes in lipid levels in human immunodeficiency disease- (HIV-) infected patients undergoing highly active antiretroviral treatment (HAART) and their association with diabetes mellitus (DM) and thyroid dysfunction. We observed changes in the levels of total cholesterol (TC) and total triglyceride (TG) of 63 HIV-infected patients in the 6 years from starting HAART and analyzed correlations between relevant parameters. TC levels of patients with normal baseline TC levels as well as those diagnosed with DM or impaired fasting glucose (IFG) increased significantly (P  < 0.05) as did the TG levels of patients with normal baseline TG levels (P  < 0.05). TC levels of patients with hypercholesterolemia in the year HAART was initiated were significantly higher than those of patients with normal baseline TC levels (P  < 0.05) for all 6 years. TC levels of patients diagnosed with DM were significantly higher than those with euglycemia (P  < 0.05) 2 and 4 years after HAART commencement. Levels of TC, high-density lipoprotein-cholesterol (HDL-C), and low-density lipoprotein-cholesterol (LDL-C) were correlated negatively with viral load, whereas levels of TC and very-low-density lipoprotein-cholesterol (VLDL-C) were correlated positively with CD4+ cell counts before HAART commencement. Linear mixed-effect model demonstrated disturbance of glucose metabolism and HAART containing nevirapine and CD4+ cell count were positively correlated with TC levels after HAART commencement. These findings suggest that there are changes in the lipid levels of patients undergoing HAART, with the potential risk of dyslipidemia.

Purpose. Serum cytokines/chemokines play important roles in cryptococcal meningitis, but it is unclear whether cytokines/chemokines in cerebrospinal fluid (CSF) contribute to high intracranial pressure (HICP) in HIV-associated cryptococcal meningitis (HCM). Methods. CSF cytokines/chemokines were assayed in 17 HIV-uninfected patients, 26 HIV-infected patients without CNS infection, and 39 HCM patients at admission. Principal component analysis and correlation and logistic regression analyses were used to assess the relationships between these parameters. Results. The CSF Th1, Th2, and macrophage cytokines showed an obvious increase in HCM patients as compared to the HIV-uninfected patients and HIV-infected patients without CNS infection. CSF IL-6, GM-CSF, and IL-8 were positively correlated with CSF fungal burden. Serum CD4 count, CSF Th1 cytokines (TNF-α, TNF-β, IL-12, IL-1β, and INF-α2), and Th2 cytokines (IL-5 and IL-6) were closely related to cryptococcal meningitis. Furthermore, both Th1 cytokines (TNF-α, TNF-β, INF-γ, and IL-12) and Th2 cytokines (IL-4 and IL-10) contribute to HICP. Conclusion. Overall, the present findings indicated that both pro- and anti-inflammatory cytokines of Th1, Th2, and macrophage origin contributed to the development of HCM. Specifically, the chemokine and cytokine cascade caused by skewing of the Th1-Th2 balance and reduced CD4 count were found to be important contributors to HICP. Summary. Our research suggested that chemokine and cytokine cascade caused by skewing of the Th1-Th2 balance in HIV-infected patients played more important role than Cryptococcus numbers and size in CSF on the development of high intracranial pressure in HIV-associated cryptococcal meningitis, providing a new understanding of mechanisms of HCM.

In recent years, transmitted drug resistance (TDR) has adversely impacted upon first-line therapy for HIV-infected individuals. To understand the current subtype distribution and TDR level in Zhejiang, China we performed phylogenetic analysis and genotypic drug resistance testing of treatment-naive HIV-infected individuals. A total of 153 HIV-1 Pol genes were successfully amplified. The distribution of HIV-1 genotypes was as follows: CRF01_AE (43.8%); CRF07_BC (37.9%); subtype B/B' (7.2%); CRF08_BC (5.2%); and others (5.9%). Drug resistance analysis demonstrated that 11.1% of isolates contained at least one NRTI or NNRTI resistance-associated mutations while 2.0% were identified to be resistant to PIs. These findings enhance our understanding of the genetic diversity of HIV-1 strains circulating in Zhejiang and provide some guidelines for HIV initial treatment therapy.

The roles of cytokines and chemokines in HIV-associated cryptococcal meningitis (HCM) and HIV-associated tuberculous meningitis (HTBM) are debatable. In sum, 34 HIV-infected patients without meningitis, 44 HCM patients and 27 HTBM patients were enrolled for study. The concentrations of 22 cytokines/chemokines in cerebrospinal fluid (CSF) were assayed at admission. Principal component analysis (PCA), Pearson's and logistic regression analyses were used to assess the role of cytokines/chemokines in HCM and HTBM. We found the levels of T helper (Th)17, Th1 [interleukin (IL)-12p40, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and TNF-β and Th2 (IL-2/4/5/6/10)] cytokines were elevated in patients with meningitis compared with those in HIV-infected patients without central nervous system (CNS) infection. Furthermore, the IL-1Ra, IL-12p40, IL-17α and monocyte chemotactic protein-1 (MCP-1) levels were higher in HCM patients, while the IFN-γ, regulated upon activation, normal T cell expressed and secreted (RANTES) and interferoninducible protein-10 (IP)-10 levels were higher in HTBM patients. Elevated CSF concentrations of IL-17a, TNF-β, IL-5, IL-12p40 and IL-1Rα were closely related to meningitis, but elevated IP-10, MCP-1, RANTES and IFN-γ levels and CSF white blood cells (WBCs) were protective factors against HCM. Our study suggested that HIV-infected patients with low CSF WBCs have a high risk of HCM. Th1, Th2 and Th17 cytokines/chemokines mediate differences in the pathogenesis of HCM and TBM. Overexpressed proinflammatory MCP-1, RANTES, IFN-γ and IP-10 in CSF are protective factors against HCM but not HTBM.

Viral DNA integrated in host cells is a major barrier to completely curing HIV-1. However, genome editing using the recently developed technique of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has the potential to eradicate HIV-1. The present study aimed to use a lentiviral vector-based CRISPR/Cas9 system combined with dual-small/single guide RNAs (sgRNAs) to attack HIV-1 DNA in the latency reactivation model J-Lat 10.6 cell line and to assess off-target effects using whole-genome sequencing (WGS). We designed 12 sgRNAs targeting HIV-1 DNA, and selected high-efficiency sgRNAs for further pairwise combinations after a preliminary evaluation of the editing efficiency. Three combinations of dual-sgRNAs/Cas9 with high editing efficiency were screened successfully from multiple combinations. Among these combinations, the incidences of insertions and deletions in the sgRNA-targeted regions reached 76% and above, and no credible off-target sites were detected using WGS. The results provided comprehensive basic experimental evidence and methodological recommendations for future personalized HIV-1 treatment using CRISPR/Cas9 genome editing technology.

The prevalence of HIV-1 is increasing rapidly among the population of men who have sex with men (MSM) in China. Here, we report a novel HIV type 1 recombinant form consisting of CRF01_AE and CRF07_BC detected in a male patient through homosexual behavior. A phylogenic analysis revealed that this unique recombinant form (URF) exhibits a complex genomic structure with three CRF07_BC regions inserted into the CRF01_AE backbone. Recently, several second-generation recombinant forms (e.g., CRF01_AE/CRF07_BC) have been identified among MSM in China. The emergence of such URFs highlights the complexity of the HIV-1 epidemic among this population. Therefore, further molecular epidemiological investigation is required to track the genetic evolution of HIV-1 strains.

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