Developing a Human Cytomegalovirus strain for better in vitro research

Investigations into Human Cytomegalovirus (HCMV) pathogenesis should be based on strains that closely reflect the causative agent of disease, however HCMV invariably mutates in vitro, generating phenotypically distinct laboratory-adapted strains. In particular, mutations are selected in the HCMV genome UL128 locus (UL128L) that encodes sub-units of a virion envelope pentameric complex that impedes virus propagation in fibroblasts (the cell type most commonly used in vitro), but is required for infection of several naturally targeted cell-types (e.g. epithelial, endothelial, and myeloid cells). Addressing this issue, the genome of wildtype HCMV strain Merlin was cloned as a stable bacterial artificial chromosome (BAC), however similarly to clinical isolates, viruses reconstituted from the Merlin-BAC grow poorly and are prone to de novo mutation in cell culture. Direct comparison to viruses from the Merlin-BAC revealed that viruses from the BACcloned versions of strains TR (TR-BAC), TB40E (TB40-BAC4) and VR1814 (FIX-BAC) could be propagated more efficiently in fibroblasts, despite containing intact UL128L ORFs. Unique nucleotide variations identified in TB40-BAC4 and FIX-BAC UL128L ORFs were transferred into the Merlin-BAC, generating variants that produced greater titres of cell-free virus following reconstitution. Virions from these novel Merlin variants displayed reduced pentameric complex content, but remained able to infect epithelial cells, albeit with slightly compromised efficiency. The greater fitness of viruses from these novel Merlin-BAC variants alleviated the selective pressures for the selection of de novo UL128L mutations in fibroblasts. The Merlin virion proteome was determined, with up to 30 novel components identified to provide a more comprehensive picture of wildtype HCMV virion composition. Comparison of virions from several Merlin variants demonstrated that varying pentameric complex content impacted the incorporation of other components, however virions from the novel Merlin variants produced in this work closely matched those from the parental Merlin variant containing wildtype UL128L ORFs. Thus, the novel Merlin-BAC variants produced in this work represent valuable reagents for future HCMV research

Human cytomegalovirus: taking the strain

In celebrating the 60th anniversary of the first isolation of human cytomegalovirus (HCMV), we reflect on the merits and limitations of the viral strains currently being used to develop urgently needed treatments. HCMV research has been dependent for decades on the high-passage strains AD169 and Towne, heavily exploiting their capacity to replicate efficiently in fibroblasts. However, the genetic integrity of these strains is so severely compromised that great caution needs to be exercised when considering their past and future use. It is now evident that wild-type HCMV strains are not readily propagated in vitro. HCMV mutants are rapidly selected during isolation in fibroblasts, reproducibly affecting gene RL13, the UL128 locus (which includes genes UL128, UL130 and UL131A) and often the UL/b′ region. As a result, the virus becomes less cell associated, altered in tropism and less pathogenic. This problem is not restricted to high-passage strains, as even low-passage strains can harbour biologically significant mutations. Cloning and manipulation of the HCMV genome as a bacterial artificial chromosome (BAC) offers a means of working with stable, genetically defined strains. To this end, the low-passage strain Merlin genome was cloned as a BAC and sequentially repaired to match the viral sequence in the original clinical sample from which Merlin was derived. Restoration of UL128L to wild type was detrimental to growth in fibroblasts, whereas restoration of RL13 impaired growth in all cell types tested. Stable propagation of phenotypically wild-type virus could be achieved only by placing both regions under conditional expression. In addition to the development of these tools, the Merlin transcriptome and proteome have been characterized in unparalleled detail. Although Merlin may be representative of the clinical agent, high-throughput whole-genome deep sequencing studies have highlighted the remarkable high level of interstrain variation present in circulating virus. There is a need to develop systems capable of addressing the significance of this diversity, free from the confounding effects of genetic changes associated with in vitro adaptation. The generation of a set of BAC clones, each containing the genome of a different HCMV strain repaired to match the sequence in the clinical sample, would provide a pathway to address the biological and clinical effects of natural variation in wild-type HCMV

Genetic stability of BAC-deerived human cytomegalovirus during culture in vitro

Clinical human cytomegalovirus (HCMV) strains invariably mutate when propagated in vitro. Mutations in gene RL13 are selected in all cell types, whereas in fibroblasts mutants in the UL128 locus (UL128L; genes UL128, UL130, and UL131A) are also selected. In addition, sporadic mutations are selected elsewhere in the genome in all cell types. We sought to investigate conditions under which HCMV can be propagated without incurring genetic defects. Bacterial artificial chromosomes (BACs) provide a stable, genetically defined source of viral genome. Viruses were generated from BACs containing the genomes of strains TR, TB40, FIX, and Merlin, as well as from Merlin-BAC recombinants containing variant nucleotides in UL128L from TB40-BAC4 or FIX-BAC. Propagation of viruses derived from TR-BAC, TB40-BAC4, and FIX-BAC in either fibroblast or epithelial cells was associated with the generation of defects around the prokaryotic vector, which is retained in the unique short (US) region of viruses. This was not observed for Merlin-BAC, from which the vector is excised in derived viruses; however, propagation in epithelial cells was consistently associated with mutations in the unique long b′ (UL/b′) region, all impacting on gene UL141. Viruses derived from Merlin-BAC in fibroblasts had mutations in UL128L, but mutations occurred less frequently with recombinants containing UL128L nucleotides from TB40-BAC4 or FIX-BAC. Viruses derived from a Merlin-BAC derivative in which RL13 and UL128L were either mutated or repressed were remarkably stable in fibroblasts. Thus, HCMV containing a wild-type gene complement can be generated in vitro by deriving virus from a self-excising BAC in fibroblasts and repressing RL13 and UL128L. IMPORTANCE Researchers should aim to study viruses that accurately represent the causative agents of disease. This is problematic for HCMV because clinical strains mutate rapidly when propagated in vitro, becoming less cell associated, altered in tropism, more susceptible to natural killer cells, and less pathogenic. Following isolation from clinical material, HCMV genomes can be stabilized by cloning into bacterial artificial chromosomes (BACs), and then virus is regenerated by DNA transfection. However, mutations can occur not only during isolation prior to BAC cloning but also when virus is regenerated. We have identified conditions under which BAC-derived viruses containing an intact, wild-type genome can be propagated in vitro with minimal risk of mutants being selected, enabling studies of viruses expressing the gene complement of a clinical strain. However, even under these optimized conditions, sporadic mutations can occur, highlighting the advisability of sequencing the HCMV stocks used in experiments

The pentameric complex drives immunologically covert cell -cell transmission of wild-type human cytomegalovirus

Human cytomegalovirus (HCMV) strains that have been passaged in vitro rapidly acquire mutations that impact viral growth. These laboratory-adapted strains of HCMV generally exhibit restricted tropism, produce high levels of cell-free virus, and develop susceptibility to natural killer cells. To permit experimentation with a virus that retained a clinically relevant phenotype, we reconstructed a wild-type (WT) HCMV genome using bacterial artificial chromosome technology. Like clinical virus, this genome proved to be unstable in cell culture; however, propagation of intact virus was achieved by placing the RL13 and UL128 genes under conditional expression. In this study, we show that WT-HCMV produces extremely low titers of cell-free virus but can efficiently infect fibroblasts, epithelial, monocyte-derived dendritic, and Langerhans cells via direct cell–cell transmission. This process of cell–cell transfer required the UL128 locus, but not the RL13 gene, and was significantly less vulnerable to the disruptive effects of IFN, cellular restriction factors, and neutralizing antibodies compared with cell-free entry. Resistance to neutralizing antibodies was dependent on high-level expression of the pentameric gH/gL/gpUL128–131A complex, a feature of WT but not passaged strains of HCMV

Temporal development and neutralising potential of antibodies against SARS-CoV-2 in hospitalised COVID-19 patients: An observational cohort study

Antibody responses are important in the control of viral respiratory infection in the human host. What is not clear for SARS-CoV-2 is how rapidly this response occurs, or when antibodies with protective capability evolve. Hence, defining the events of SARS-CoV-2 seroconversion and the time frame for the development of antibodies with protective potential may help to explain the different clinical presentations of COVID-19. Furthermore, accurate descriptions of seroconversion are needed to inform the best use of serological assays for diagnostic testing and serosurveillance studies. Here, we describe the humoral responses in a cohort of hospitalised COVID-19 patients (n = 19) shortly following the onset of symptoms. Commercial and ‘in-house’ serological assays were used to measure IgG antibodies against different SARS-CoV-2 structural antigens–Spike (S) S1 sub-unit and Nucleocapsid protein (NP)–and to assess the potential for virus neutralisation mediated specifically by inhibition of binding between the viral attachment protein (S protein) and cognate receptor (ACE-2). Antibody response kinetics varied amongst the cohort, with patients seroconverting within 1 week, between 1–2 weeks, or after 2 weeks, following symptom onset. Anti-NP IgG responses were generally detected earlier, but reached maximum levels slower, than anti-S1 IgG responses. The earliest IgG antibodies produced by all patients included those that recognised the S protein receptor-binding domain (RBD) and were capable of inhibiting binding to ACE-2. These data revealed events and patterns of SARS-CoV-2 seroconversion that may be important predictors of the outcome of infection and guide the delivery of clinical services in the COVID-19 response

Suppression of costimulation by human cytomegalovirus promotes evasion of cellular immune defenses.

CD58 is an adhesion molecule that is known to play a critical role in costimulation of effector cells and is intrinsic to immune synapse structure. Herein, we describe a virally encoded gene that inhibits CD58 surface expression. Human cytomegalovirus (HCMV) UL148 was necessary and sufficient to promote intracellular retention of CD58 during HCMV infection. Blocking studies with antagonistic anti-CD58 mAb and an HCMV UL148 deletion mutant (HCMV∆UL148) with restored CD58 expression demonstrated that the CD2/CD58 axis was essential for the recognition of HCMV-infected targets by CD8+ HCMV-specific cytotoxic T lymphocytes (CTLs). Further, challenge of peripheral blood mononuclear cells ex vivo with HCMV∆UL148 increased both CTL and natural killer (NK) cell degranulation against HCMV-infected cells, including NK-driven antibody-dependent cellular cytotoxicity, showing that UL148 is a modulator of the function of multiple effector cell subsets. Our data stress the effect of HCMV immune evasion functions on shaping the immune response, highlighting the capacity for their potential use in modulating immunity during the development of anti-HCMV vaccines and HCMV-based vaccine vectors

Quantitative temporal viromics: an approach to investigate host-pathogen interaction

A systematic quantitative analysis of temporal changes in host and viral proteins throughout the course of a productive infection could provide dynamic insights into virus-host interaction. We developed a proteomic technique called “quantitative temporal viromics” (QTV), which employs multiplexed tandem-mass-tag-based mass spectrometry. Human cytomegalovirus (HCMV) is not only an important pathogen but a paradigm of viral immune evasion. QTV detailed how HCMV orchestrates the expression of >8,000 cellular proteins, including 1,200 cell-surface proteins to manipulate signaling pathways and counterintrinsic, innate, and adaptive immune defenses. QTV predicted natural killer and T cell ligands, as well as 29 viral proteins present at the cell surface, potential therapeutic targets. Temporal profiles of >80% of HCMV canonical genes and 14 noncanonical HCMV open reading frames were defined. QTV is a powerful method that can yield important insights into viral infection and is applicable to any virus with a robust in vitro model