Antibody responses to SARS-CoV-2 can be detected in most infected individuals 10-15 d after the onset of COVID-19 symptoms. However, due to the recent emergence of SARS-CoV-2 in the human population, it is not known how long antibody responses will be maintained or whether they will provide protection from reinfection. Using sequential serum samples collected up to 94 d post onset of symptoms (POS) from 65 individuals with real-time quantitative PCR-confirmed SARS-CoV-2 infection, we show seroconversion (immunoglobulin (Ig)M, IgA, IgG) in >95% of cases and neutralizing antibody responses when sampled beyond 8 d POS. We show that the kinetics of the neutralizing antibody response is typical of an acute viral infection, with declining neutralizing antibody titres observed after an initial peak, and that the magnitude of this peak is dependent on disease severity. Although some individuals with high peak infective dose (ID 50 > 10,000) maintained neutralizing antibody titres >1,000 at >60 d POS, some with lower peak ID 50 had neutralizing antibody titres approaching baseline within the follow-up period. A similar decline in neutralizing antibody titres was observed in a cohort of 31 seropositive healthcare workers. The present study has important implications when considering widespread serological testing and antibody protection against reinfection with SARS-CoV-2, and may suggest that vaccine boosters are required to provide long-lasting protection.

Summary SARS-CoV-2 Spike protein is critical for virus infection via engagement of ACE2 1 , and is a major antibody target. Here we report chronic SARS-CoV-2 with reduced sensitivity to neutralising antibodies in an immune suppressed individual treated with convalescent plasma, generating whole genome ultradeep sequences over 23 time points spanning 101 days. Little change was observed in the overall viral population structure following two courses of remdesivir over the first 57 days. However, following convalescent plasma therapy we observed large, dynamic virus population shifts, with the emergence of a dominant viral strain bearing D796H in S2 and ΔH69/ΔV70 in the S1 N-terminal domain NTD of the Spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype diminished in frequency, before returning during a final, unsuccessful course of convalescent plasma. In vitro , the Spike escape double mutant bearing ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, whilst maintaining infectivity similar to wild type. D796H appeared to be the main contributor to decreased susceptibility but incurred an infectivity defect. The ΔH69/ΔV70 single mutant had two-fold higher infectivity compared to wild type, possibly compensating for the reduced infectivity of D796H. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy associated with emergence of viral variants with evidence of reduced susceptibility to neutralising antibodies.

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Antibody (Ab) responses to SARS-CoV-2 can be detected in most infected individuals 10-15 days following the onset of COVID-19 symptoms. However, due to the recent emergence of this virus in the human population it is not yet known how long these Ab responses will be maintained or whether they will provide protection from re-infection. Using sequential serum samples collected up to 94 days post onset of symptoms (POS) from 65 RT-qPCR confirmed SARS-CoV-2-infected individuals, we show seroconversion in >95% of cases and neutralizing antibody (nAb) responses when sampled beyond 8 days POS. We demonstrate that the magnitude of the nAb response is dependent upon the disease severity, but this does not affect the kinetics of the nAb response. Declining nAb titres were observed during the follow up period. Whilst some individuals with high peak ID50 (>10,000) maintained titres >1,000 at >60 days POS, some with lower peak ID50 had titres approaching baseline within the follow up period. A similar decline in nAb titres was also observed in a cohort of seropositive healthcare workers from Guy′s and St Thomas′ Hospitals. We suggest that this transient nAb response is a feature shared by both a SARS-CoV-2 infection that causes low disease severity and the circulating seasonal coronaviruses that are associated with common colds. This study has important implications when considering widespread serological testing, Ab protection against re-infection with SARS-CoV-2 and the durability of vaccine protection.

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.HSV ͉ latency-associated transcript ͉ latency ͉ reactivation ͉ human H erpes simplex virus 1 and 2 (HSV-1 and HSV-2) are closely related human herpes viruses. HSV-1 and HSV-2 establish lifelong incurable latency in and reactivate preferentially from trigeminal ganglia and dorsal root ganglia to cause oro-facial and genital herpes, respectively. Although infections are usually mild, these viruses can cause severe disease including encephalitis and neonatal herpes, and HSV-2 infection is a risk factor for HIV acquisition. The only readily detectable viral transcript during latency of both HSV-1 and HSV-2 is the noncoding latency-associated transcript (LAT), which is transcribed from within the long repeats of the viral genome ( Fig. 1) (1). A Ϸ8-kb primary LAT is spliced, yielding a stable Ϸ2-kb LAT intron (2). Deletion of the LAT promoter in both HSV-1 and HSV-2 reduces the efficiency of reactivation (3-5), and substitution of HSV-1 LAT for native HSV-2 LAT sequences confers an HSV-1 reactivation phenotype (6). The HSV-1 LAT is currently believed to act in part by increasing the establishment or maintenance of latency, likely via an effect on the survival of acutely infected neurons (7), which may be mediated by inhibition of apoptosis in infected neurons (8). The molecular function of HSV-2 LAT remains largely unknown.miRNAs are a family of 21Ϸ24-nt noncoding RNAs that regulate gene expression based on sequence similarity to their targets. Mammalian viruses including EBV, Kaposi's sarcoma-associated herpesvirus, human cytomegalovirus, and SV40 encode viral miRNAs (9). Viral encoded miRNAs were predicted for HSV-1 (10, 11) and also for HSV-2 (10). However, no miRNAs have been identified in HSV-2 or HSV-1 LAT sequences. Results HSV-2 LAT Exon 2 Encodes a miRNA.To find miRNAs within the HSV-2 LAT region, a plasmid containing the LAT sequence and its promoter (pSSK) and a mutant plasmid expressing LAT under control of the CMV-IE promoter (pCMV-SSK) were transfected into 293 cells. Small RNAs isolated from the transfected cells were cloned and sequenced. A 22-23-nt HSV-2 RNA sequence (designated HSV-2 miR-I) appeared at a frequenc...

We recently identified an acutely and latently expressed viral microRNA (miRNA), miR-I, encoded by herpes simplex virus 2 (HSV-2) latency-associated transcript (LAT) through small RNA cloning and two miRNAs encoded by HSV-1 LAT through prediction. We now report the use of high-throughput sequencing technology to identify two additional relatively less-abundant viral miRNAs, miR-II and miR-III, encoded by HSV-2 LAT exon 2. miR-II includes two miRNAs, miR-II-5p and miR-II-3p, which are processed from the same miRNA precursor. miR-II and miR-III map antisense to the 5 untranslated region of ICP34.5 and to the coding region of ICP0 exon 3, respectively. These novel miRNAs are conserved in different HSV-2 strains, and their presence in infected-and transfected-cell cultures was confirmed by Northern hybridization. All three HSV-2 LAT-encoded miRNAs map to genome locations similar to those of three out of four identified HSV-1 LAT-encoded miRNAs, but the sequences of these miRNAs are not conserved. The expression of LAT-encoded miRNAs is negatively regulated by ICP4, the major viral transactivator. We further show that, similar to miR-I, miR-II is able to efficiently silence the expression of ICP34.5, a key viral neurovirulence factor, and that miR-III is able to silence the expression of ICP0, a key viral transactivator. All these data suggest that LAT sequences likely contribute to HSV latency and reactivation through tight control of these LAT-encoded miRNAs and their viral targets.Herpes simplex virus 1 (HSV-1) and HSV-2 are closely related herpesviruses. HSV-1 typically infects the facial region and establishes a lifelong latent infection in sensory neurons of the trigeminal ganglia, while HSV-2 typically infects the genital region and establishes a lifelong latent infection in sensory neurons of the sacral dorsal root ganglia. Periodically, either virus may reactivate to cause symptomatic or asymptomatic recurrences in the area served by these sensory neurons. HSV-2 and HSV-1 have similar latent transcription patterns, in which the latency-associated transcript (LAT) is transcribed from within the genomic long repeats. In contrast to other viral promoters, the LAT promoter is highly active during latency, and LAT is the only viral gene product that is readily detectable during latency (39). HSV-1 LAT expression is inhibited by ICP4, the major viral transactivator required for most post-␣ gene expression (9, 12, 24), through an ICP4 binding site near the LAT transcription initiation site (14). The LAT introns (ϳ2.2 kb in HSV-2 and ϳ2 kb and 1.4 kb in HSV-1), which overlap the ICP0 transcript in an antisense direction, are much more abundant and stable than the ϳ8.5-kbp primary LAT transcript (13), which overlaps both the ICP0 and ICP34.5 transcripts in an antisense direction. ICP0 can transactivate a number of viral and host genes and is essential for HSV reactivation (4,5,18,19). ICP34.5, a key viral neurovirulence factor, is a protein kinase R inhibitor and is required for efficient viral replication in neurons...

CRABS CLAW (CRC), a member of the YABBY gene family, is required for nectary and carpel development. To further understand CRC regulation in Arabidopsis thaliana, we performed phylogenetic footprinting analyses of 59 upstream regions of CRC orthologs from three Brassicaceae species, including Arabidopsis. Phylogenetic footprinting efficiently identified functionally important regulatory regions (modules), indicating that CRC expression is regulated by a combination of positive and negative regulatory elements in the modules. Within the conserved modules, we identified putative binding sites of LEAFY and MADS box proteins, and functional in vivo analyses revealed their importance for CRC expression. Both expression and genetic studies demonstrate that potential binding sites for MADS box proteins within the conserved regions are functionally significant for the transcriptional regulation of CRC in nectaries. We propose that in wild-type flowers, a combination of floral homeotic gene activities, specifically the B class genes APETALA3 and PISTILLATA and the C class gene AGAMOUS act redundantly with each other and in combination with SEPALLATA genes to activate CRC in the nectaries and carpels. In the absence of B and C class gene activities, other genes such as SHATTERPROOF1/2 can substitute if they are ectopically expressed, as in an A class mutant background (apetala2). These MADS box proteins may provide general floral factors that must work in conjunction with specific factors in the activation of CRC in the nectaries and carpels.

A chlorhexidine-based surface antiseptic protocol can interrupt transmission of MRSA in the intensive care unit, but strains carrying qacA/B genes may be unaffected or potentially spread more rapidly.