Improved Production of HIV-1 Subtype C Protease from Transgenic E. Coli

Background: Human Immunodeficiency Virus 1 (HIV-1) subtype C is responsible for the majority of infections of patients in Southern Africa. The HIV protease is a primary target for the development of highly efficient anti-retroviral pharmaceuticals because of its pivotal role in the maturation of the virus in the host cell. For target validation of novel HIV protease inhibitors, there is a need for the availability of an abundance of this protease. Objective: This study reports an optimized method to produce HIV-1 protease derived from HIV-1 subtype C. Methods: It involves the use of a transgenic E. coli strain that overexpresses the native form of the enzyme via inclusion bodies. A stringent method for the isolation, purification, and renaturation resulted in the production of highly pure active HIV-1 protease. In order to facilitate an increase in protease yields, an optimized growth strategy was developed. In this regard, a chemically defined medium with lower glucose content and devoid of essential amino acids of the TCA cycle was used as an alternative to the widely used nutrient-rich Luria Bertani (LB) medium. Results: Results indicated an increase in protease yield up to twice the amount, thereby making this medium an attractive alternative for increasing biomass and HIV protease production for future research. Conclusion: An optimized method for HIV-1 protease derived from HIV-1 subtype C production using chemically defined media was established. This was achieved using a known method to isolate and purify the enzyme with the use of a specialized feeding strategy.

Epidemiology and Transmitted HIV-1 Drug Resistance among Treatment-Naïve Individuals in Israel, 2010–2018

Despite the low prevalence of HIV-1 in Israel, continuous waves of immigration may have impacted the local epidemic. We characterized all people diagnosed with HIV-1 in Israel in 2010–2018. The demographics and clinical data of all individuals (n = 3639) newly diagnosed with HIV-1 were retrieved. Subtypes, transmitted drug-resistance mutations (TDRM), and phylogenetic relations, were determined in >50% of them. In 39.1%, HIV-1 transmission was through heterosexual contact; 34.3% were men who have sex with men (MSM); and 10.4% were people who inject drugs. Many (>65%) were immigrants. Israeli-born individuals were mostly (78.3%) MSM, whereas only 9% of those born in Sub-Saharan Africa (SSA), Eastern Europe and Central Asia (EEU/CA), were MSM. The proportion of individuals from SSA decreased through the years 2010–2018 (21.1% in 2010–2012; 16.8% in 2016–2018) whereas those from EEU/CA increased significantly (21% in 2010–2012; 27.8% in 2016–2018, p < 0.001). TDRM were identified in 12.1%; 3.7, 3.3 and 6.6% had protease inhibitors (PI), nucleotide reverse transcriptase inhibitors (NRTI), and non-nucleoside reverse transcriptase inhibitors (NNRTI) TDRM, respectively, with the overall proportion remaining stable in the studied years. None had integrase TDRM. Subtype B was present in 43.9%, subtype A in 25.2% (A6 in 22.8 and A1 in 2.4%) and subtype C in 17.1% of individuals. Most MSM had subtype B. Subtype C carriers formed small clusters (with one unexpected MSM cluster), A1 formed a cluster mainly of locally-born patients with NNRTI mutations, and A6 formed a looser cluster of individuals mainly from EEU. Israelis, <50 years old, carrying A1, had the highest risk for having TDRM. In conclusion, an increase in immigrants from EEU/CA and a decrease in those from SSA characterized the HIV-1 epidemic in 2010–2018. Baseline resistance testing should still be recommended to identify TDRM, and improve surveillance and care.

Potential Associations of Mutations within the HIV-1 Env and Gag Genes Conferring Protease Inhibitor (PI) Drug Resistance

An increasing number of patients in Africa are experiencing virological failure on a second-line antiretroviral protease inhibitor (PI)-containing regimen, even without resistance-associated mutations in the protease region, suggesting a potential role of other genes in PI resistance. Here, we investigated the prevalence of mutations associated with Lopinavir/Ritonavir (LPV/r) failure in the Envelope gene and the possible coevolution with mutations within the Gag-protease (gag-PR) region. Env and Gag-PR sequences generated from 24 HIV-1 subtype C infected patients failing an LPV/r inclusive treatment regimen and 344 subtype C drug-naïve isolates downloaded from the Los Alamos Database were analyzed. Fisher’s exact test was used to determine the differences in mutation frequency. Bayesian network probability was applied to determine the relationship between mutations occurring within the env and gag-PR regions and LPV/r treatment. Thirty-five mutations in the env region had significantly higher frequencies in LPV/r-treated patients. A combination of Env and Gag-PR mutations was associated with a potential pathway to LPV/r resistance. While Env mutations were not directly associated with LPV/r resistance, they may exert pressure through the Gag and minor PR mutation pathways. Further investigations using site-directed mutagenesis are needed to determine the impact of Env mutations alone and in combination with Gag-PR mutations on viral fitness and LPV/r efficacy.

Molecular Identification and Characterization of a Genotype 3 Hepatitis E Virus (HEV) Strain Detected in a Wolf Faecal Sample, Italy

Hepatitis E virus (HEV) infection is a major health problem worldwide. In developed countries, zoonotic transmission of HEV genotypes (Gt) 3 and 4 is caused by the ingestion of raw or undercooked meat of infected pigs and wild boars, the main reservoirs of HEV. However, additional animals may harbour HEV or HEV-related strains, including carnivores. In this study, we investigated the molecular epidemiology of orthohepeviruses in wild canids by screening a total of 136 archival faecal samples, collected from wolves (42) and red foxes (94) in Northwestern Italy. Orthohepevirus RNA was identified in a faecal specimen, collected from a wolf carcass in the province of La Spezia (Liguria Region, Italy). The nearly full-length (7212 nucleotides) genome of the strain HEV/81236/Wolf/2019/ITA (GenBank accession no. MZ463196) was determined by combining a sequence-independent single-primer amplification (SISPA) approach with the Oxford Nanopore Technologies sequencing platform. Upon phylogenetic analysis, the HEV detected in wolf was segregated into clade HEV-3.1, displaying the highest nucleotide (nt) identity (89.0–93.3%) to Gt3 strains belonging to subtype c. Interestingly, the wolf faecal sample also contained porcine astrovirus sequences, endorsing the hypothesis of a dietary origin of the HEV strain due to preying habits.

Evolutionary dynamics of HIV-1 subtype C in Brazil

The extensive genetic diversity of HIV-1 is a major challenge for the prevention and treatment of HIV-1 infections. Subtype C accounts for most of the HIV-1 infections in the world but has been mainly localized in Southern Africa, Ethiopia and India. For elusive reasons, South Brazil harbors the largest HIV-1 subtype C epidemic in the American continent that is elsewhere dominated by subtype B. To investigate this topic, we collected clinical data and viral sequences from 2611 treatment-naïve patients diagnosed with HIV-1 in Brazil. Molecular epidemiology analysis supported 35 well-delimited transmission clusters of subtype C highlighting transmission within South Brazil but also from the South to all other Brazilian regions and internationally. Individuals infected with subtype C had lower probability to be deficient in CD4+ T cells when compared to subtype B. The HIV-1 epidemics in the South was characterized by high female-to-male infection ratios and women-to-child transmission. Our results suggest that HIV-1 subtype C probably takes advantage of longer asymptomatic periods to maximize transmission and is unlikely to outcompete subtype B in settings where the infection of women is relatively less relevant. This study contributes to elucidate factors possibly underlying the geographical distribution and expansion patterns of the most spread HIV-1 subtypes.

The Evolution of Regulatory Elements in the Emerging Promoter-Variant Strains of HIV-1 Subtype C

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not the coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but no significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a coinfection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.

A Molecular Study on Hepatitis E Virus (HEV) in Pigs in Bulgaria

Information on hepatitis E virus (HEV) strains circulating in animal reservoirs in Bulgaria is currently lacking. Herein, by screening HEV seropositive sera obtained from Bulgarian swine and wild boars, viral RNA was detected at high prevalence rate (28.2%) in industrial pigs. Sequence analysis of the partial polymerase (RdRp) region revealed the highest genetic correlation with HEVs of genotype (Gt) 3 identified in French and Dutch patients. For three such strains, a 700-bp fragment of the open reading frame 2 gene was generated. On phylogenetic analysis, the Bulgarian strains clustered tightly (93.8–98.3% nt) with human and animal HEVs classified within the Gt3 subtype c.

Assessment of an LSDV-Vectored Vaccine for Heterologous Prime-Boost Immunizations against HIV

The modest protective effects of the RV144 HIV-1 vaccine trial have prompted the further exploration of improved poxvirus vector systems that can yield better immune responses and protection. In this study, a recombinant lumpy skin disease virus (LSDV) expressing HIV-1 CAP256.SU gp150 (Env) and a subtype C mosaic Gag was constructed (LSDVGC5) and compared to the equivalent recombinant modified vaccinia Ankara (MVAGC5). In vitro characterization confirmed that cells infected with recombinant LSDV produced Gag virus-like particles containing Env, and that Env expressed on the surface of the cells infected with LSDV was in a native-like conformation. This candidate HIV-1 vaccine (L) was tested in a rabbit model using different heterologous vaccination regimens, in combination with DNA (D) and MVA (M) vectors expressing the equivalent HIV-1 antigens. The four different vaccination regimens (DDMMLL, DDMLML, DDLMLM, and DDLLMM) all elicited high titers of binding and Tier 1A neutralizing antibodies (NAbs), and some regimens induced Tier 1B NAbs. Furthermore, two rabbits in the DDLMLM group developed low levels of autologous Tier 2 NAbs. The humoral immune responses elicited against HIV-1 Env by the recombinant LSDVGC5 were comparable to those induced by MVAGC5.

Not all wavelengths are created equal: disinfection of SARS-CoV-2 using UVC radiation is wavelength-dependent

SARS-CoV-2 is mostly transmitted through close contact with infected people by infected aerosols and fomites. Ultraviolet subtype C (UVC) lamps and light-emitting diodes can be used to disrupt the transmission chain by disinfecting fomites, thus managing the disease outbreak progression. Here, we assess the ultraviolet wavelengths that are most effective in inactivation of SARS-CoV-2 on fomites. Variations in UVC wavelengths impact the dose required for disinfection of SARS-CoV-2 and alter how rapidly and effectively disruption of the virus transmission chain can be achieved. This study reveals that shorter wavelengths (254–268 nm) take a maximum of 6.25 mJ/cm2 over 5 s to obtain a target SARS-CoV-2 reduction of 99.9%. Longer wavelengths, like 280 nm, take longer irradiation time and higher dose to inactivate SARS-CoV-2. These observations emphasize that SARS-CoV-2 inactivation is wavelength-dependent.

Quantifying the Impact of Ultraviolet Subtype C in Reducing Airborne Pathogen Transmission and Improving Energy Efficiency in Healthy Buildings: A Kahn–Mariita Equivalent Ventilation Model

There is growing evidence that viruses responsible for pandemics, such as Middle East respiratory syndrome and severe acute respiratory syndrome, are mainly spread through aerosols. Recommendations have been introduced to reduce the transmission risks of virulent airborne viral particles by increasing ventilation rates, expressed in air changes per hour (ACHs), effectively improving the dilution of pathogens via mechanical ventilation. However, infrastructural and operational costs associated with upgrades of building heating, ventilation, and air conditioning systems make these solutions expensive. It is well documented that Ultraviolet Subtype C (UVC) disinfection can help lower exposure risks by inactivating viruses and the performance of such solutions can translate into equivalent ventilation. Here, we present the first framework to extract the optimal UVC requirements to improve facility management yet ensuring compliance with ventilation guidelines at lower energy costs. The Kahn–Mariita (KM) model considers the air quality of shared enclosed spaces over time by supplementing the existing mechanical ventilation with localized UVC air treatment and includes variables such as room size, occupancy, existing ventilation, and target equivalent ACH. For example, the model applied to a conference room shows that a UVC chamber with recirculation rates of 160 m3/h increases ventilation from an ACH 3 to 7.9 and reduces the room’s reset time from 46 to &lt;10 min with as little as 1 W. Recirculation rates of 30 m3/h however offer no benefits beyond 200 mW, with an eACH of 3.9 and reset time of 31 min. The first finding is that single-pass disinfection is not an appropriate metric of performance, i.e., low recirculation rates increase single-pass disinfection, and, however, only treats a portion of the space volume within a given time, limiting the overall performance. Conversely, higher recirculation rates decrease single-pass disinfection but treat larger portions of air, potentially multiple times, and are therefore expected to lower the transmission risk faster. The second result is that for fixed amounts of recirculating air flow, increasing UVC power helps with diminishing return, while for a fixed UVC power, increasing the recirculating air flow will always help. This dynamic is particularly important toward optimizing solutions, given the constraints system engineers must work with, and particularly to design for end-user benefits such as increased occupancy, in-dwelling time, or reduction of shared-space reset time.