Background: HCV infection is very common in multi-transfused β-thalassemia patients who need regular blood transfusions. Aim: The study was conducted to determine the epidemiology of HCV in multi-transfused β-thalassemia patients in West Bengal, India. Methods: Over a span of six years, blood samples were collected from HCV sero-reactive β-thalassemia patients and processed for viral RNA isolation followed by nested RT-PCR for qualitative viremia detection. The HCV genotype was determined by amplifying the partial HCV core gene by nested RT-PCR followed by DNA sequencing and NCBI genotyping tools. Phylogenetic and phylogeographic studies were performed with MEGA-X and BEAST software, respectively. Results: Out of 917 multi-transfused HCV sero-reactive β-thalassemia patients, 598 (65.21%) were HCV RNA positive while 250 (41.80%) had spontaneously cleared the virus. A significant percentage of male patients from rural areas (p = 0.042) and economically backward class (p = 0.002) were at higher risk of HCV infection. Female thalassemia patients and individuals belonging to ages 11–15 years had higher chances of spontaneous clearance. The most prevalent circulatory HCV genotype was 3a (78.26%) followed by 1b (12.04%). Phylogeographic analyses revealed that the 3a strains share genomic similarities with strains from Pakistan, Sri Lanka, and Thailand, whereas the 1b strains share similarities with strains from Thailand, Vietnam, Russia, and China. Uncommon HCV subtypes 3g and 3i were also detected. Conclusion: The high prevalence of HCV infection among β-thalassemia patients of West Bengal, India indicates NAT-based assays should be implemented for HCV screening in donor blood to eliminate HCV by 2030.

Summary: Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.

Mammalian orthoreovirus (MRV) infection induces phosphorylation of translation initiation factor eIF2α which promotes formation of discrete cytoplasmic inclusions, termed stress granules (SGs). SGs are emerging as a component of the innate immune response to virus infection, and modulation of SG assembly is a common mechanism employed by viruses to counter this antiviral response. We previously showed that MRV infection induces SGs early, then interferes with SG formation as infection proceeds. In this work, we found that SG associated proteins localized to the periphery of virus-encoded cytoplasmic structures, termed virus factories (VFs), where viral transcription, translation, and replication occur. The localization of SG proteins to VFs was dependent on polysome dissociation and occurred via association of SG effector protein, G3BP1, with MRV non-structural protein σNS, which localizes to VFs via association with VF nucleating protein, μNS. Deletion analysis of the σNS RNA binding domain and G3BP1 RNA (RRM) and ribosomal (RGG) binding domains showed that the association and VF localization of G3BP1 is not occurring solely through RNA or ribosomal binding, but requires both RNA and ribosomal binding domains of G3BP1 for maximal VFL localization and σNS association. Co-expression of σNS and μNS resulted in disruption of normal SG puncta, and in cells lacking G3BP1, MRV replication was enhanced in a manner correlating with strain-dependent induction of host translation shutoff. These results suggest that σNS association with and relocalization of G3BP1 to the VF periphery plays a role in SG disruption to facilitate MRV replication in the host translational shutoff environment. IMPORTANCE: SGs and SG effector proteins have emerged as important, yet poorly understood, players in the host’s innate immune response to virus infection. MRV infection induces SGs early during infection that are dispersed and/or prevented from forming during late stages of infection despite continued activation of the eIF2α signaling pathway. Cellular and viral components involved in disruption of SGs during late stages of MRV infection remain to be elucidated. This work provides evidence that MRV disruption of SGs may be facilitated by association of MRV non-structural protein σNS with major SG effector protein G3BP1 and subsequent localization of G3BP1 and other SG associated proteins around the periphery of virus encoded factories, interrupting the normal formation of SGs. Our findings also reveal the importance of G3BP1 as an inhibitor of MRV replication during infection for the first time.

Within infected host cells, mammalian orthoreovirus (MRV) forms viral factories (VFs) which are sites of viral transcription, translation, assembly, and replication. MRV non-structural protein, μNS, comprises the structural matrix of VFs and is involved in recruiting other viral proteins to VF structures. Previous attempts have been made to visualize VF dynamics in live cells but due to current limitations in recovery of replicating reoviruses carrying large fluorescent protein tags, researchers have been unable to directly assess VF dynamics from virus-produced μNS. We set out to develop a method to overcome this obstacle by utilizing the 6 amino-acid (CCPGCC) tetracysteine (TC)-tag and FlAsH-EDT2 reagent. The TC-tag was introduced into eight sites throughout μNS, and the capacity of the TC-μNS fusion proteins to form virus factory-like (VFL) structures and colocalize with virus proteins was characterized. Insertion of the TC-tag interfered with recombinant virus rescue in six of the eight mutants, likely as a result of loss of VF formation or important virus protein interactions. However, two recombinant (r)TC-μNS viruses were rescued and VF formation, colocalization with associating virus proteins, and characterization of virus replication were subsequently examined. Furthermore the rTC-μNS viruses were utilized to infect cells and examine VF dynamics using live cell microscopy. These experiments demonstrate active VF movement with fusion events as well as transient interactions between individual VFs, and demonstrate the importance of microtubule stability for VF fusion during MRV infection. This work provides important groundwork for future in depth studies of VF dynamics and host cell interactions. Importance: MRV has historically been used as a model to study the double-stranded RNA (dsRNA) Reoviridae family, which infect and cause disease in humans, animals, and plants. During infection, MRV forms VFs that play a critical role in virus infection, but remain to be fully characterized. To study VFs, researchers have focused on visualizing the non-structural protein μNS which forms the VF matrix. This work provides the first evidence of recovery of replicating reoviruses in which VFs can be labeled in live cells via introduction of a TC-tag into the μNS open reading frame. Characterization of each recombinant reovirus sheds light on μNS interactions with viral proteins. Moreover, utilizing the TC labeling FlAsH-EDT2 biarsenical reagent to visualize VFs, evidence is provided of dynamic VF movement and interactions at least partially dependent on intact microtubules.

Background: Post transfusion acquired HCV infection is common in high-risk group individuals such as multi-transfused β-thalassemia patients who depend on regular blood transfusions. This study was conducted to determine epidemiology and distribution of HCV in multi-transfused β-thalassemia patients, in West Bengal, India. Methods: Over a span of six years blood samples were collected from HCV sero-reactive β-thalassemic patients and processed for viral RNA isolation followed by nested RT-PCR for qualitative viremia detection. HCV genotype was determined by amplifying partial HCV core gene by nested RT-PCR, DNA sequencing and using NCBI genotyping tools. Phylogenetic and phylogeographic studies were performed with online MEGA-X and BEAST 1.10.0 software respectively. Results: Out of 917 multi-transfused HCV sero-reactive β-thalassemic patients, 598 (65.21%) were positive for HCV RNA while 250 (41.80%) had spontaneously cleared the virus. Female thalassemic patients and individuals belonging to ages 10-14 years had higher chances of spontaneous clearance. The most prevalent circulatory HCV genotype was 3a (78.11%) followed by 1b (12.20%). Phylogeographic analyses revealed that the 3a strains share similarity with Pakistan, Sri Lanka and Thailand whereas the 1b strains share similarity with Thailand, Vietnam, Russia and China. Conclusion: The prevalence of HCV infection is very high among Indian β-thalassemic patients, necessitates a critical look into the prevailing transfusion practices and requires implementation of more rigid donor screening criteria to decrease the rate of transfusion transmitted HCV infection, especially in multi-transfused thalassemic patients. The use of more sensitive NAT based assays for HCV detection in donor blood is a compressing need of the hour.