Hepatitis C virus (HCV) is a major cause of liver disease worldwide. To establish and maintain chronic infection, HCV extensively rearranges cellular organelles to generate distinct compartments for viral RNA replication and virion assembly. Here, we review our current knowledge of how HCV proliferates and remodels ER-derived membranes while preserving and expanding associated lipid droplets during viral infection. Unraveling the molecular mechanisms responsible for HCV-induced membrane reorganization will enhance our understanding of the HCV life-cycle, the associated liver pathology, and the biology of the ER:lipid droplet interface in general.

Background

HIV prevention in India has mostly focussed on heterosexual transmission. Data on homosexual transmission are not readily available from India. We therefore assessed the probability of acquiring and transmitting HIV for men who sell sex to men and compared this with women who sell sex in India.

Methods

Sexual behaviour characteristics of 6661 men who have sex with men and 6648 women who sell sex were obtained in the Indian state of Andhra Pradesh through confidential interviews. These, along with estimates of HIV rates among them and risk of HIV transmission per unprotected sex act from other sources, were used to calculate their annual probability of acquiring and transmitting HIV.

Results

Of 6661 men who have sex with men in this sample, 1776 (26.7%) had sold sex to men. For every 1000 men who sell sex to men, annually 146 (95% confidence interval [CI] 116-179) would acquire HIV and HIV would be transmitted to 55 (95% CI 42-71) men who do not sell sex or women. These estimates were higher by 6.7 (95% CI 4.9-9.2) times for acquiring HIV and 2.5 (95% CI 2.0-3.2) times for transmitting HIV to sex partners outside their group, as compared with similar estimates for women who sell sex. In this sample, the average annual probability of acquiring HIV was higher among men who have sex with men but do not sell sex as compared with women who sell sex.

Conclusion

These data indicate that men who sell sex to men are at much higher risk of acquiring and transmitting HIV than women who sell sex. Therefore, men who sell sex to men and their clients warrant substantial attention for comprehensive HIV prevention in India.

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells.

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.

Highlights

We developed a novel panel of 7 quantitative RT-ddPCRs assays for SARS-Cov-2 Our panel targets nongenic and genic regions in genomic and subgenomic RNAs All assays detect 1-10 copies and are linear over 3-4 orders of magnitude All assays correlated with the clinical Abbott SARS-CoV-2 Viral Load Assay Clinical samples showed higher copy numbers for targets at the 3’ end of the genome

A hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of sensitive, quantitative RT-ddPCR assays designed to target regions spanning the genome of SARS-CoV-2. Our assays target untranslated regions (5', 3') as well as different coding regions, including non-structural genes that are only found in full length (genomic) RNA and structural genes that are found in genomic as well as different subgenomic RNAs. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 gene expression and may also inform the development of improved diagnostic tools and therapeutics.

This paper provides a detailed overview and discussion of anaesthesia in patients with mucopolysaccharidosis (MPS), the evaluation of risk factors in these patients and their anaesthetic management, including emergency airway issues. MPS represents a group of rare lysosomal storage disorders associated with an array of clinical manifestations. The high prevalence of airway obstruction and restrictive pulmonary disease in combination with cardiovascular manifestations poses a high anaesthetic risk to these patients. Typical anaesthetic problems include airway obstruction after induction or extubation, intubation difficulties or failure [can't intubate, can't ventilate (CICV)], possible emergency tracheostomy and cardiovascular and cervical spine issues. Because of the high anaesthetic risk, the benefits of a procedure in patients with MPS should always be balanced against the associated risks. Therefore, careful evaluation of anaesthetic risk factors should be made before the procedure, involving evaluation of airways and cardiorespiratory and cervical spine problems. In addition, information on the specific type of MPS, prior history of anaesthesia, presence of cervical instability and range of motion of the temporomandibular joint are important and may be pivotal to prevent complications during anaesthesia. Knowledge of these risk factors allows the anaesthetist to anticipate potential problems that may arise during or after the procedure. Anaesthesia in MPS patients should be preferably done by an experienced (paediatric) anaesthetist, supported by a multidisciplinary team (ear, nose, throat surgeon and intensive care team), with access to all necessary equipment and support.

The long-lasting COVID-19 pandemic and increasing SARS-CoV-2 variants demand effective drugs for prophylactics and treatment. Protein-based biologics offer high specificity, yet their noncovalent interactions often lead to drug dissociation and incomplete inhibition. Here, we have developed covalent nanobodies capable of binding with SARS-CoV-2 irreversibly via a proximity-enabled reactive therapeutic (PERx) mechanism. A latent bioreactive amino acid (FFY) was designed and genetically encoded into nanobodies to accelerate the PERx reaction rate. Compared with the noncovalent wild-type nanobody, the FFY-incorporated covalent nanobodies neutralized both wild-type SARS-CoV-2 and its Alpha, Delta, Epsilon, Lambda, and Omicron variants with drastically higher potency. This PERx-enabled covalent-nanobody strategy and the related insights into increased potency can be valuable to developing effective therapeutics for various viral infections.

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3' end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment.

Background

The majority of sex work in India is clandestine due to unfavorable legal environment and discrimination against female sex workers (FSWs). We report data on who these women are and when they get involved with sex work that could assist in increasing the reach of HIV prevention activities for them.

Methods

Detailed documentation of demography and various aspects of sex work was done through confidential interviews of 6648 FSWs in 13 districts in the Indian state of Andhra Pradesh. The demography of FSWs was compared with that of women in the general population.

Results

A total of 5010 (75.4%), 1499 (22.5%), and 139 (2.1%) street-, home-, and brothel-based FSWs, respectively, participated. Comparison with women of Andhra Pradesh revealed that the proportion of those aged 20-34 years (75.6%), belonging to scheduled caste (35.3%) and scheduled tribe (10.5%), illiterate (74.7%), and of those separated/divorced (30.7%) was higher among FSWs (p < 0.001). The FSWs engaged in sex work for >5 years were more likely to be non-street-based FSWs, illiterate, living in small urban towns, and to have started sex work between 12-15 years of age. The mean age at starting sex work (21.7 years) and gap between the first vaginal intercourse and the first sexual intercourse in exchange for money (6.6 years) was lower for FSWs in the rural areas as compared with those in large urban areas (23.9 years and 8.8 years, respectively).

Conclusion

These data highlight that women struggling with illiteracy, lower social status, and less economic opportunities are especially vulnerable to being infected by HIV, as sex work may be one of the few options available to them to earn money. Recommendations for actions are made for long-term impact on reducing the numbers of women being infected by HIV in addition to the current HIV prevention efforts in India.

Hepatitis C virus (HCV) is a leading cause of liver disease, but insight into virus-host interactions remains limited. We systematically used affinity purification/mass spectrometry to define the host interactions of all ten HCV proteins in hepatoma cells. We combined these studies with RNAi knockdown of corresponding genes using a two-step scoring approach to generate a map of 139 high-confidence HCV-host protein-protein interactions. We found mitochondrial proteins highly involved in HCV infection and characterized an interaction between the viral core protein and host protein within bgcn homolog (WIBG). Expression of core prevents WIBG from binding its regular interaction partners Y14 and Magoh, two known mediators of the nonsense-mediated mRNA decay pathway. We discovered that this surveillance pathway is disrupted in HCV-infected cells, causing potentially harmful transcripts to accumulate. Our study provides a comprehensive view of HCV-host interactions and uncovers mechanisms for how HCV perturbs host functions during infection.