Antiviral lectins from red and blue-green algae show potent in vitro and in vivo activity against hepatitis C virus.

Hepatitis C virus (HCV) infection is a significant public health problem with over 170,000,000 chronic carriers and infection rates increasing worldwide. Chronic HCV infection is one of the leading causes of hepatocellular carcinoma which was estimated to result in ∼10,000 deaths in the United States in the year 2011. Current treatment options for HCV infection are limited to PEG-ylated interferon alpha (IFN-α), the nucleoside ribavirin and the recently approved HCV protease inhibitors telaprevir and boceprevir. Although showing significantly improved efficacy over the previous therapies, treatment with protease inhibitors has been shown to result in the rapid emergence of drug-resistant virus. Here we report the activity of two proteins, originally isolated from natural product extracts, which demonstrate low or sub-nanomolar in vitro activity against both genotype I and genotype II HCV. These proteins inhibit viral infectivity, binding to the HCV envelope glycoproteins E1 and E2 and block viral entry into human hepatocytes. In addition, we demonstrate that the most potent of these agents, the protein griffithsin, is readily bioavailable after subcutaneous injection and shows significant in vivo efficacy in reducing HCV viral titers in a mouse model system with engrafted human hepatocytes. These results indicate that HCV viral entry inhibitors can be an effective component of anti-HCV therapy and that these proteins should be studied further for their therapeutic potential.

Reconstructing the epidemic history of HIV-1 circulating recombinant forms CRF07_BC and CRF08_BC in East Asia: the relevance of genetic diversity and phylodynamics for vaccine strategies.

HIV-1 CRF07_BC and CRF08_BC are closely related circulating recombinant forms (CRFs) with serious public health consequences in China. The temporal and spatial dynamics of these CRFs were determined by estimating their times of divergence, using phylogenetic and Bayesian coalescent methods. Studies of the timelines of CRF07_BC and CRF08_BC trace the expansion of these strains back their origins to Yunnan province. The present study highlights the relevance of incorporating evolutionary and molecular epidemiological analyses into an in-depth understanding of the genesis of HIV epidemic, providing information for determining regional and global public health policies, including future vaccine strategies.

Explosive HIV-1 subtype B' epidemics in Asia driven by geographic and risk group founder events.

We explored the timescale, spatial spread, and risk group population structure of HIV-1 subtype B', the cause of explosive blood-borne HIV-1 epidemics among injecting drug users (IDUs) and former plasma donors (FPDs) in Asia. Sequences from FPDs in China formed a distinct monophyletic cluster within subtype B'. Further analysis revealed that subtype B' was founded by a single lineage of pandemic subtype B around 1985. Subsequently, the FPD cluster appears to have derived from a single subtype B' lineage around 1991, corroborating the hypothesis that FPD outbreaks stemmed from the preceding epidemic among IDUs in Southeast Asia, most likely from the Golden-Triangle region.

Identification of a novel second-generation circulating recombinant form (CRF48_01B) in Malaysia: a descendant of the previously identified CRF33_01B.

A molecular epidemiological investigation conducted among injecting drug users in eastern Peninsular Malaysia in 2007 identified a cluster of sequences (n = 3) located outside any known HIV-1 genotype. Analyses of near full-length nucleotide sequences of these strains from individuals with no recognizable linkage revealed that they have an identical subtype structure comprised of CRF01_AE and subtype B', distinct from any known circulating recombinant forms (CRFs). This novel CRF, designated CRF48_01B, is closely related to CRF33_01B, previously identified in Kuala Lumpur. Phylogenetic analysis of multiple CRF48_01B genome regions showed that CRF48_01B forms a monophyletic cluster within CRF33_01B, suggesting that this new recombinant is very likely a descendant of CRF33_01B. CRF48_01B thus represents one of the first examples of a "second-generation" CRF, generated by additional crossover with pre-existing CRFs. Corroborating these results, Bayesian molecular clock analyses indicated that CRF48_01B emerged in approximately 2001, approximately approximately 8 years after the emergence of CRF33_01B.

[Recent advances in HIV vaccine research and development--newly unearthed challenges].

Historically, vaccines have been among the most effective public health interventions, preventing the spread of viral infections. However, HIV vaccine has thus far been extremely elusive. The recent failure of the first T-cell vaccine has led to a reexamination of the direction of HIV-vaccine field. An empirical approach that has been successfully applied to many human viruses appears not effective for HIV. Re-examination has pointed to a need to emphasize fundamental questions of HIV vaccine discovery. In particular, recent advances in the study on the earliest phase of HIV-1 infection unearthed novel challenges for HIV vaccine development.

Isolation and characterization of a replication-competent molecular clone of an HIV-1 circulating recombinant form (CRF33_01B).

A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs) have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1), a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5' LTR (long terminal repeat) region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone), showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4+, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.

Subtype-specific differences in the human immunodeficiency virus type 1 reverse transcriptase connection subdomain of CRF01_AE are associated with higher levels of resistance to 3'-azido-3'-deoxythymidine.

We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3'-azido-3'-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.

Phylodynamic analysis of the dissemination of HIV-1 CRF01_AE in Vietnam.

To estimate the epidemic history of HIV-1 CRF01_AE in Vietnam and adjacent Guangxi, China, we determined near full-length nucleotide sequences of CRF01_AE from a total of 33 specimens collected in 1997-1998 from different geographic regions and risk populations in Vietnam. Phylogenetic and Bayesian molecular clock analyses were performed to estimate the date of origin of CRF01_AE lineages. Our study reconstructs the timescale of CRF01_AE expansion in Vietnam and neighboring regions and suggests that the series of CRF01_AE epidemics in Vietnam arose by the sequential introduction of founder strains into new locations and risk groups. CRF01_AE appears to have been present among heterosexuals in South-Vietnam for more than a decade prior to its epidemic spread in the early 1990s. In the late 1980s, the virus spread to IDUs in Southern Vietnam and subsequently in the mid-1990s to IDUs further north. Our results indicate the northward dissemination of CRF01_AE during this time.

Chronology of the HIV-1 CRF07_BC expansion in East Asia.

The HIV-1 epidemic among injecting drug users (IDU) in Taiwan is caused primarily by CRF07_BC infections. Evolutionary analyses, which utilize outgroup reference strains from northwestern China (Xinjiang), reveal that CRF07_BC was introduced into southern Taiwan in 1998-2001 and spread to central-northern Taiwan in 2001-2003, causing the largest HIV/AIDS epidemic in Taiwan. The separate introduction of CRF07_BC into Xinjiang occurred in 1992-1995. This study illustrates the temporal dynamics of CRF07_BC spread among IDU across east Asia.