Multiple Amino Acid Substitutions Research Articles

Genomic Evolution and Surveillance of Respiratory Syncytial Virus during the 2023-2024 Season.

Respiratory syncytial virus (RSV) is a significant cause of morbidity, particularly in infants. This study describes RSV genomic diversity and disease outcomes during the 2023-2024 season in the Johns Hopkins Hospital System (JHHS). Between August and December 2023, 406 patient samples were sequenced, showing that RSV-B GB5.0.5a was the dominant genotype detected. RSV-A genotype GA2.3.5 was detected less frequently. Metadata analysis of patient data revealed that, although RSV-B was more commonly detected, patients with RSV-A infections were more frequently hospitalized. Analysis of both the G- and F-genes revealed multiple amino acid substitutions in both RSV-A and RSV-B, with some positions within the F-protein that could be associated with evasion of antibody responses. Phylogenetic analysis revealed the genetic diversity of circulating GB5.0.5a and GA2.3.5 genotypes. This study serves as an important baseline for genomic surveillance of RSV within the JHHS and will assist in characterizing the impact of the newly approved RSV vaccines on RSV genomic evolution and the emergence of escape mutations.

H5N1 high pathogenicity avian influenza virus in migratory birds exhibiting low pathogenicity in mallards increases its risk of transmission and spread in poultry

In 2020, an H5N1 avian influenza virus of clade 2.3.4.4b was detected in Europe for the first time and was spread throughout the world by wild migratory birds, resulting in the culling of an unprecedented number of wild birds and poultry due to the epidemic. In February 2023, we isolated and identified a strain of H5N1 high pathogenicity avian influenza virus from a swab sample from a grey crane in Ningxia, China. Phylogenetic analysis of the Hemagglutinin (HA) gene showed that the virus belonged to clade 2.3.4.4b, and several gene segments were closely related to H5N1 viruses infecting humans in China. Analysis of key amino acid sites revealed that the virus contained multiple amino acid substitutions that facilitate enhanced viral replication and mammalian pathogenicity. The results of animal challenge experiments showed that the virus is highly pathogenic to chickens, moderately pathogenic to BALB/c mice, and highly infectious but not lethal to mallards. Moreover, the virus exhibited minor antigenic drift compared with the H5-Re14 vaccine strain. To this end, we need to pay more attention to the monitoring of wild birds to prevent further spread of viruses to poultry and mammals, including humans.

Prevalence of occult hepatitis B infection among blood donors in Saint Petersburg

The aim of this study was to assess the prevalence of occult hepatitis B infection among blood donors in St. Petersburg, as well as to characterize the identified virus isolates. The study material was represented by 2800 blood plasma samples collected in 2019 from blood donors living in St. Petersburg. The ELISA study for HBV marker rate consisted of HBsAg, anti-HBs IgG, anti-HBcore IgG. HBV DNA was analyzed by nested PCR with real-time hybridization-fluorescence detection on three targets allowing to determine virus DNA at low viral load, including HBsAg-negative chronic hepatitis B. Hepatitis B serological markers were detected in 69.43% of those surveyed, HBsAg was found in 0.43% of individuals, and all of which donated blood first time. A significant excess of the anti-HBcore IgG antibodies occurrence among primary donors (15.1%) compared with repeated/regular donors (7.48%) was shown. The prevalence of virus DNA in the group was 3.14%, including 2.71% of cases in HBsAg-negative CHB. Based on phylogenetic analysis of 88 isolates, HBV subgenotypes were determined in the following order: D1 and D2, 40.91% each, D3 and A2, 9.09% each. While determining the serological subtype in detected isolates, the serotype ayw3 (52.27%) vs ayw2 (46.59%) and adw2 (10.23%) prevailed. Drug resistance mutations, including compensatory ones, were detected in six examined patients (6.82%). In all genotype D isolates, multiple amino acid substitutions were identified in the RT, SHB, MHB, LHB, and Core regions; mutations in the preCore region were detected in 21.59% samples. In the MHR of the HBV genotype D genome, twenty-six positions were identified in which amino acid substitutions occurred, and all isolates showed modifications at positions 113, 114, 131, 134, 159, 161, 168, in 76 — at position 122, in 68 — at position 127, in 36 — at position 118, in 24 — at position 128. In HBV A2 isolates, mutations T113S, S143T, Y161F were identified. Nine isolates in the preCore region showed a polymorphism including a stop codon W28*W; in five isolates the W28S substitution was shown in the same position, and the W28*S variant was found in one more sample. The high incidence of HBsAg-negative CHB cases among blood donors, as well as the predominance of HBV isolates that simultaneously carry mutations resulting in diagnostic failure of HBsAg tests and prophylactic failure of immunoglobulin or vaccines and virus reactivation, mutations that contribute to disease progression obviously pose a threat to health and require to be further examined.

The intrinsically disordered transcriptional activation domain of CIITA is functionally tuneable by single substitutions: An exception or a new paradigm?

During protein evolution, some amino acid substitutions modulate protein function ("tuneability"). In most proteins, the tuneable range is wide and can be sampled by a set of protein variants that each contains multiple amino acid substitutions. In other proteins, the full tuneable range can be accessed by a set of variants that each contains a single substitution. Indeed, in some globular proteins, the full tuneable range can be accessed by the set of site-saturating substitutions at an individual "rheostat" position. However, in proteins with intrinsically disordered regions (IDRs), most functional studies-which would also detect tuneability-used multiple substitutions or small deletions. In disordered transcriptional activation domains (ADs), studies with multiple substitutions led to the "acidic exposure" model, which does not anticipate the existence of rheostat positions. In the few studies that did assess effects of single substitutions on AD function, results were mixed: the ADs of two full-length transcription factors did not show tuneability, whereas a fragment of a third AD was tuneable by single substitutions. In this study, we tested tuneability in the AD of full-length human class II transactivator (CIITA). Sequence analyses and experiments showed that CIITA's AD is an IDR. Functional assays of singly-substituted AD variants showed that CIITA's function was highly tuneable, with outcomes not predicted by the acidic exposure model. Four tested positions showed rheostat behavior for transcriptional activation. Thus, tuneability of different IDRs can vary widely. Future studies are needed to illuminate the biophysical features that govern whether an IDR is tuneable by single substitutions.

Genome characterization of influenza A and B viruses in New South Wales, Australia, in 2019: A retrospective study using high-throughput whole genome sequencing.

During the 2019 severe influenza season, New South Wales (NSW) experienced the highest number of cases in Australia. This study retrospectively investigated the genetic characteristics of influenza viruses circulating in NSW in 2019 and identified genetic markers related to antiviral resistance and potential virulence. The complete genomes of influenza A and B viruses were amplified using reverse transcription-polymerase chain reaction (PCR) and sequenced with an Illumina MiSeq platform. When comparing the sequencing data with the vaccine strains and reference sequences, the phylogenetic analysis revealed that most NSW A/H3N2 viruses (n= 68; 94%) belonged to 3C.2a1b and a minority (n= 4; 6%) belonged to 3C.3a. These viruses all diverged from the vaccine strain A/Switzerland/8060/2017. All A/H1N1pdm09 viruses (n= 20) showed genetic dissimilarity from vaccine strain A/Michigan/45/2015, with subclades 6B.1A.5 and 6B.1A.2 identified. All B/Victoria-lineage viruses (n= 21) aligned with clade V1A.3, presenting triple amino acid deletions at positions 162-164 in the hemagglutinin protein, significantly diverging from the vaccine strain B/Colorado/06/2017. Multiple amino acid substitutions were also found in the internal proteins of influenza viruses, some of which have been previously reported in hospitalized influenza patients in Thailand. Notably, the oseltamivir-resistant marker H275Y was present in one immunocompromised patient infected with A/H1N1pdm09 and the resistance-related mutation I222V was detected in another A/H3N2-infected patient. Considering antigenic drift and the constant evolution of circulating A and B strains, we believe continuous monitoring of influenza viruses in NSW via the high-throughput sequencing approach provides timely and pivotal information for both public health surveillance and clinical treatment.

Whole Genomic Constellation of Avian Reovirus Strains Isolated from Broilers with Arthritis in North Carolina, USA.

Avian reovirus (ARV) is an emerging pathogen which causes significant economic challenges to the chicken and turkey industry in the USA and globally, yet the molecular characterization of most ARV strains is restricted to a single particular gene, the sigma C gene. The genome of arthrogenic reovirus field isolates (R18-37308 and R18-38167), isolated from broiler chickens in North Carolina (NC), USA in 2018, was sequenced using long-read next-generation sequencing (NGS). The isolates were genotyped based on the amino acid sequence of sigma C (σC) followed by phylogenetic and amino acid analyses of the other 11 genomically encoded proteins for whole genomic constellation and genetic variation detection. The genomic length of the NC field strains was 23,494 bp, with 10 dsRNA segments ranging from 3959 bp (L1) to 1192 bp (S4), and the 5' and 3' untranslated regions (UTRs) of all the segments were found to be conserved. R18-37308 and R18-38167 were found to belong to genotype (G) VI based on the σC analysis and showed nucleotide and amino acid sequence identity ranging from 84.91-98.47% and 83.43-98.46%, respectively, with G VI strains. Phylogenetic analyses of individual genes of the NC strains did not define a single common ancestor among the available completely sequenced ARV strains. Nevertheless, most sequences supported the Chinese strain LY383 as a probable ancestor of these isolates. Moreover, amino acid analysis revealed multiple amino acid substitution events along the entirety of the genes, some of which were unique to each strain, which suggests significant divergence owing to the accumulation of point mutations. All genes from R18-37308 and R18-38167 were found to be clustered within genotypic clusters that included only ARVs of chicken origin, which negates the possibility of genetic pooling or host variation. Collectively, this study revealed sequence divergence between the NC field strains and reference ARV strains, including the currently used vaccine strains could help updating the vaccination regime through the inclusion of these highly divergent circulating indigenous field isolates.

Current Update on New Delhi Metallo-β-lactamase (NDM) Variants: New Challenges in the Journey of Evolution.

New Delhi Metallo-β-lactamase is an enzyme produced by gram-negative bacteria which has become one of the global concerns for physicians to treating the infection. These Metallo- β-lactamase are capable of catalyzing the hydrolysis of almost all β-lactam antibiotics, endangering infection treatment. Substitution of single or multiple amino acids results in new NDM variants. Forty NDM variants have been identified in different bacterial strains across the globe. In this review, we focused on the structural insight of all NDM variants including the type of amino acid residues and their position of substitution, country of origin, and type of bacteria carrying these resistant markers. We also discussed the carbapenemase activity and stability of enzymes that helps to design potent inhibitors to combat drug-resistant infections.

Mutations of SARS-CoV-2 Structural Proteins in the Alpha, Beta, Gamma, and Delta Variants: Bioinformatics Analysis.

COVID-19 and Middle East Respiratory Syndrome are two pandemic respiratory diseases caused by coronavirus species. The novel disease COVID-19 caused by SARS-CoV-2 was first reported in Wuhan, Hubei Province, China, in December 2019, and became a pandemic within 2-3 months, affecting social and economic platforms worldwide. Despite the rapid development of vaccines, there have been obstacles to their distribution, including a lack of fundamental resources, poor immunization, and manual vaccine replication. Several variants of the original Wuhan strain have emerged in the last 3 years, which can pose a further challenge for control and vaccine development. The aim of this study was to comprehensively analyze mutations in SARS-CoV-2 variants of concern (VoCs) using a bioinformatics approach toward identifying novel mutations that may be helpful in developing new vaccines by targeting these sites. Reference sequences of the SARS-CoV-2 spike (YP_009724390) and nucleocapsid (YP_009724397) proteins were compared to retrieved sequences of isolates of four VoCs from 14 countries for mutational and evolutionary analyses. Multiple sequence alignment was performed and phylogenetic trees were constructed by the neighbor-joining method with 1000 bootstrap replicates using MEGA (version 6). Mutations in amino acid sequences were analyzed using the MultAlin online tool (version 5.4.1). Among the four VoCs, a total of 143 nonsynonymous mutations and 8 deletions were identified in the spike and nucleocapsid proteins. Multiple sequence alignment and amino acid substitution analysis revealed new mutations, including G72W, M2101I, L139F, 209-211 deletion, G212S, P199L, P67S, I292T, and substitutions with unknown amino acid replacement, reported in Egypt (MW533289), the United Kingdom (MT906649), and other regions. The variants B.1.1.7 (Alpha variant) and B.1.617.2 (Delta variant), characterized by higher transmissibility and lethality, harbored the amino acid substitutions D614G, R203K, and G204R with higher prevalence rates in most sequences. Phylogenetic analysis among the novel SARS-CoV-2 variant proteins and some previously reported β-coronavirus proteins indicated that either the evolutionary clade was weakly supported or not supported at all by the β-coronavirus species. This study could contribute toward gaining a better understanding of the basic nature of SARS-CoV-2 and its four major variants. The numerous novel mutations detected could also provide a better understanding of VoCs and help in identifying suitable mutations for vaccine targets. Moreover, these data offer evidence for new types of mutations in VoCs, which will provide insight into the epidemiology of SARS-CoV-2.

Variations in NSP1 of Porcine Reproductive and Respiratory Syndrome Virus Isolated in China from 1996 to 2022.

Since its successful isolation in China in 1995, the porcine reproductive and respiratory syndrome virus (PRRSV) has been mutating into highly pathogenic strains by constantly changing pathogenicity and genetic makeup. In this study, we investigated the prevalence and genetic variation of nonstructural protein 1 (NSP1) in PRRSV-2, the main strain prevalent in China. After formulating hypotheses regarding the biology of the NSP1 protein, the nucleotide and amino acid similarity of NSP1 were analyzed and compared in 193 PRRSV-2 strains. The results showed that NSP1 has a stable hydrophobic protein with a molecular weight of 43,060.76 Da. Although NSP1 lacked signal peptides, it could regulate host cell signaling. Furthermore, NSP1 of different strains had high nucleotide (79.6-100%) and amino acid similarity (78.6-100%). In the amino acid sequence comparison of 15 representative strains of PRRSV-2, multiple amino acid substitution sites were found in NSP1. Phylogenetic tree analysis showed that lineages 1 and 8 had different evolutionary branches with long genetic distances. This study lays the foundation for an in-depth understanding of the nature and genetic variation of NSP1 and the development of a safe and effective vaccine in the future.

Molecular characterization and phylogenetic study of the hemagglutinin-neuraminidase gene of newcastle disease virus isolated from peacock (Pavo cristatus) and Turkey (Meleagris) and its comparison with broiler isolates.

Newcastle disease has been endemic within the Iranian poultry industry for decades. However, the genetic nature of the circulating Hemagglutinin-Neuraminidase (HN) gene among Iranian domesticated bird populations is broadly unexplored. The presented study was carried out to gain insights into the biological and molecular characterization of four complete HN genes isolated from turkey, peacock, and broiler isolates in Iran between 2018 and 2020. The phylogenetic analysis revealed that the isolates belong to the Newcastle disease virus (NDV) subgenotype VII.1.1, previously known as VIIL. Further analysis demonstrated the thermostable substitutions S315P and I369V within the isolates. Finding the N-glycosylation site (NIS) at positions 144-146 and the cysteine residue 123 might influence the fusogenicity abilities of the isolates, while identification of multiple amino acid substitutions in both antigenic sites, especially I514V and E347Q, and the binding sites of the HN protein, raised concern about the pathogenicity of the isolates. In addition, the annual rate of change based on the HN gene of Iranian NDV was calculated at about 1.8088E-3 between 2011 and 2020. In conclusion, a new NDV variant with multiple site mutagenesis is circulating not only among chickens but also in turkey and captive birds such as peafowls, and failure of routine vaccination programs could be attributed to the differences between circulating NDV strains and those used in vaccine manufacturing. Therefore, future legislation aimed at providing vaster vaccination cover and biosecurity plans will be needed to control the spread of circulating NDV strains.

Abstract B018: The development of macrocyclic peptide Ras inhibitors

Abstract While there has been tremendous progress developing small molecule covalent K-Ras G12C inhibitors that bind Ras-off (GDP) and inhibit nucleotide exchange, there remains a need for additional K-Ras inhibitors with different Ras isoform/mutant selectivity profiles and Ras-on (GTP) mechanisms of inhibition. Of particular interest are compounds with a pan-K-Ras profile that inhibit multiple oncogenic mutants and wild type K-Ras, but achieve a therapeutic window by sparing H and N-Ras. In addition, addressing cancers driven by K-Ras oncogenic mutants beyond G12C, K-Ras amplification, or upstream dysregulation where K-Ras is maintained in the GTP-bound state will require “Ras-on” compounds that prevent effector binding. We were interested in the potential of peptide macrocycles that bind the Switch II pocket of K-Ras, in a different conformation than the small molecules, to inhibit Ras-on by allosterically blocking Raf and other effector binding. KRpep-2d is a 19-residue disulfide bridged peptide macrocycle discovered by phage display against K-Ras G12D bound to GDP. Due to its high molecular weight (2561 Da), polarity and metabolic instability, KRpep2d is inactive in in vitro cell assays. And while biochemical experiments have shown that it binds the Ras-off GDP-bound conformation of K-Ras with high affinity to inhibit nucleotide exchange, it has low affinity for K-Ras GTP and does not allosterically inhibit Raf binding. Using parallel macrocyclic peptide chemistry, we made 180 analogs of KRpep-2d to explore truncation of the exocyclic residues and replacement of the labile disulfide bond. UNP-6752, the best compound from this exploratory library, had K-Ras affinities similar to KRpep2d with a 50-fold K-Ras GDP/GTP preference, but was only 11 residues and cyclized with a non-reducible linker. Critically this compound lacked the eight arginine residues of KrPep-2d, which both preclude passive-cell permeability and induce mast cell degranulation. A second iteration of 350 compounds designed to explore single or multiple amino acid substitutions in the core of UNP-6752 led to compound UNP-7572 with sub-micromolar affinity for K-Ras GTP and an IC50 in a K-Ras-Raf TR-FRET assay of ~800 nM. Working from the UNP-7572 we are tuning selectivity for K-Ras and our progress further improving allosteric Ras-Raf inhibition, cell permeability and activity will be presented. Citation Format: Kristopher Josephson, Anthony Silvestri, Jennifer L. Fribourgh, Walter M. Bray, Brian David, Cameron C. Hanna, Sai Kumar Chakka, Alex Hyatt, Monique Porcella, Dio Dieppa-Matos, Cameron R. Pye, Joshua Schwochert. The development of macrocyclic peptide Ras inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B018.

Diversity of blaPOM in carbapenem-resistant opportunistic pathogenic Pseudomonas otitidis in municipal wastewater.

Metallo-β-lactamases (MBLs) encoding carbapenem resistance in wastewater are a well-known serious threat to human health. Twelve Pseudomonas otitidis isolates obtained from a municipal wastewater treatment plant (WWTP) in Hawaii were found to possess a subclass B3 MBL - POM (P. otitidis MBL), with a minimum inhibition concentration (MIC) range of 8-16 mg/L. The unrooted neighbor-joining phylogenetic tree showed that these blaPOM genes isolated in wastewater samples (n = 12) were distinctly different from other reference genes isolated from clinical, freshwater, animal, and soil samples except for isolates MR7, MR8, and MR11. MR7, MR8, and MR11 were found to have 4, 3, and 3 amino acid substitutions when compared to the type strain MC10330T and were closely clustered to the clinical reference genes. The meropenem hydrolysis experiment showed that isolates with multiple amino acid substitutions completely hydrolyzed 64 mg/L of meropenem in 7 h. The emergence of the opportunistic pathogen P. otitidis chromosomally encoding blaPOM in the treated municipal wastewater is an alarming call for the spread of this MBL in the environment. Further studies are required to understand the mechanism and regulation of this carbapenem-resistant β-lactamase in order to fill in the knowledge gap.

Enhancement in the catalytic efficiency of D-amino acid oxidase from Glutamicibacter protophormiae by multiple amino acid substitutions

D-Amino acid oxidase (DAAO) is an imperative oxidoreductase that oxidizes D-amino acids to corresponding keto acids, producing ammonia and hydrogen peroxide. Previously, based on the sequence alignment of DAAO from Glutamicibacter protophormiae (GpDAAO-1) and (GpDAAO-2), 4 residues (E115, N119, T256, T286) at the surface regions of GpDAAO-2, were subjected to site-directed mutagenesis and achieved 4 single-point mutants with enhanced catalytic efficiency (kcat/Km) compared to parental GpDAAO-2. In the present study, to further enhance the catalytic efficiency of GpDAAO-2, a total of 11 (6 double, 4 triple, and 1 quadruple-point) mutants were prepared by the different combinations of 4 single-point mutants. All mutants and wild types were overexpressed, purified and enzymatically characterized. A triple-point mutant E115A/N119D/T286A exhibited the most significant improvement in catalytic efficiency as compared to wild-type GpDAAO-1 and GpDAAO-2. Structural modeling analysis elucidated that residue Y213 in loop region C209-Y219 might act as the active-site lid for controlling substrate access, the residue K256 substituted by threonine (K256T) might change the hydrogen bonding interaction between residue Y213 and the surrounding residues, and switch the conformation of the active-site lid from the closed state to the open state, resulting in the enhancement in substrate accessibility and catalytic efficiency.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2–5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

Finding haplotypic signatures in proteins.

The nonrandom distribution of alleles of common genomic variants produces haplotypes, which are fundamental in medical and population genetic studies. Consequently, protein-coding genes with different co-occurring sets of alleles can encode different amino acid sequences: protein haplotypes. These protein haplotypes are present in biological samples and detectable by mass spectrometry, but they are not accounted for in proteomic searches. Consequently, the impact of haplotypic variation on the results of proteomic searches and the discoverability of peptides specific to haplotypes remain unknown. Here, we study how common genetic haplotypes influence the proteomic search space and investigate the possibility to match peptides containing multiple amino acid substitutions to a publicly available data set of mass spectra. We found that for 12.42% of the discoverable amino acid substitutions encoded by common haplotypes, 2 or more substitutions may co-occur in the same peptide after tryptic digestion of the protein haplotypes. We identified 352 spectra that matched to such multivariant peptides, and out of the 4,582 amino acid substitutions identified, 6.37% were covered by multivariant peptides. However, the evaluation of the reliability of these matches remains challenging, suggesting that refined error rate estimation procedures are needed for such complex proteomic searches. As these procedures become available and the ability to analyze protein haplotypes increases, we anticipate that proteomics will provide new information on the consequences of common variation, across tissues and time.

Identification, molecular characterization, and pathological features of orf virus in sheep and goats in Punjab province, Pakistan

Orf virus (ORFV) causes an acute, contagious, skin disease of sheep and goats which is economically important. The objectives of this study were to identify ORFV and to explore its pathological and phylogenetic profiles in 350 goats and 91 sheep of 14 districts of Punjab, Pakistan, from July 2020 to July 2021. Skin scrapings (total no. of samples = 441) of suspected animals were subjected to polymerase chain reactions, phylogenetic analysis, and pathological observations. The partial length of GIF/IL-2 gene (408bp) was successfully amplified in 58/441 samples. Phylogenetic analysis of GIF/IL2 gene showed that the study isolates belonged to ORFV-cluster I, together with the viruses reported in India and China. Pakistan ORFV isolates were shared 97.6-98.7% nucleotide and 97.6-100% amino acid identities with the reference strain (NC_005336). Moreover, Chinese ORFV-isolates were detected unique multiple amino acid substitutions (F11L, Q21H, D27N, I46V, N49S, N82D, D103N, S129G) with study isolates. Naturally infected animals were anorexic, emaciated, dull, and depressed. The macroscopic lesions included multifocal to coalescing, ulceration followed by proliferative papules, pustules, and crust formation on the epidermis of gums, lips, mouth commissure, muzzles, nose, and udder. Histopathological examination revealed hyperplasia, anastomosing rete ridges formation and degenerative changes, including spongiosis and vacuolation of epidermal cells. Keratinocytes exhibited eosinophilic intracytoplasmic inclusion bodies with pyknotic and karyorrhexis nuclei. This is the first report on molecular characterization of ORFV from Pakistan, with insight into its pathogenesis and comparative analysis of pathological alterations and genetic diversity between ORFV strains reported in different geographical areas.

Variations in the NSP4 gene of the type 2 porcine reproductive and respiratory syndrome virus isolated in China from 1996 to 2021.

Porcine reproductive and respiratory syndrome virus (PRRSV) has continuously mutated since its first isolation in China in 1996, leading to difficulties in infection prevention and control. Infections caused by PRRSV-2 strains are the main epidemic strains in China, as determined by phylogenetic analysis. In this study, we focused on the prevalence and genetic variations of the non-structural protein 4 (NSP4) from PRRSV-2 over the past 20years in China. The fundamental biological properties of the NSP4 were predicted, and an analysis and comparison of NSP4 homology at the nucleotide and amino acid levels was conducted using 123 PRRSV-2 strains. The predicted molecular weight of the NSP4 protein was determined to be 21.1kDa, and it was predicted to be a stable hydrophobic protein that lacks a signal peptide. NSP4 from different strains exhibited a high degree of amino acid (85.8-100%) and nucleotide sequence homology (81.0-100%). Multiple amino acid substitutions were identified in NSP4 among 15 representative PRRSV-2 strains. Phylogenetic analysis showed that the lineage 8 and 1 strains, the most prevalent strains in China, were indifferent clades with a long genetic distance. This analysis will help fully elucidate the parameters of the PRRSV NSP4 epidemic in China to lay a foundation for adequate understanding of the function of NSP4. Genetic information results from the accumulation of conserved and non-conserved sequences. The high conservation of the NSP4 gene determines the most basic life traits and functions of PRRSV. Analyzing the spatial structure of NSP4 protein and studying the genetic evolution of NSP4 not only provide the theoretical basis for how NSP4 participates in the regulation of the innate response of the host but also provide a target for genetic manipulation and a reasonable target molecule and structure for new drug molecules.

Molecular characterization and phylogenetic analysis of porcine epidemic diarrhea virus strains circulating in China from 2020 to 2021

BackgroundPorcine epidemic diarrhea virus (PEDV), an enteric coronavirus, has become the major causative agent of acute gastroenteritis in piglets since 2010 in China.ResultsIn the current study, 91 complete spike (S) gene sequences were obtained from PEDV positive samples collected from 17 provinces in China from March 2020 to March 2021. A phylogenetic analysis showed that 92.3% (84 out of 91) of the identified strains belonged to GII subtype, while 7.7% (7 out of 91) were categorized as S-INDEL like strains and grouped within GI-c clade. Based on a recombination analysis, six of S-INDEL like strains were recombinant strains originated from S-INDEL strain FR/001/2014 and virulent strain AJ1102. In addition, PEDV variant strains (CH/GDMM/202012, CH/GXDX/202010 et al) carrying novel insertions (360QGRKS364 and 1278VDVF1281) in the S protein were observed. Furthermore, the deduced amino acid sequences for the S protein showed that multiple amino acid substitutions in the antigenic epitopes in comparison with the vaccine strains.ConclusionsIn conclusion, these data provide novel molecular evidence on the epidemiology and molecular diversity of PEDV in 2020–2021. This information may help design a strategy for controlling and preventing the prevalence of PEDV variant strains in China.

Impact of antigen test target failure and testing strategies on the transmission of SARS-CoV-2 variants

Population testing remains central to COVID-19 control and surveillance, with countries increasingly using antigen tests rather than molecular tests. Here we describe a SARS-CoV-2 variant that escapes N antigen tests due to multiple disruptive amino-acid substitutions in the N protein. By fitting a multistrain compartmental model to genomic and epidemiological data, we show that widespread antigen testing in the Italian region of Veneto favored the undetected spread of the antigen-escape variant compared to the rest of Italy. We highlight novel limitations of widespread antigen testing in the absence of molecular testing for diagnostic or confirmatory purposes. Notably, we find that genomic surveillance systems which rely on antigen population testing to identify samples for sequencing will bias detection of escape antigen test variants. Together, these findings highlight the importance of retaining molecular testing for surveillance purposes, including in contexts where the use of antigen tests is widespread.

Comparative Analysis of the pIgR Gene from the Antarctic Teleost Trematomus bernacchii Reveals Distinctive Features of Cold-Adapted Notothenioidei.

The IgM and IgT classes were previously identified and characterized in the Antarctic teleost Trematomus bernacchii, a species belonging to the Perciform suborder Notothenoidei. Herein, we characterized the gene encoding the polymeric immunoglobulin receptor (pIgR) in the same species and compared it to the pIgR of multiple teleost species belonging to five perciform suborders, including 11 Antarctic and 1 non-Antarctic (Cottoperca gobio) notothenioid species, the latter living in the less-cold peri-Antarctic sea. Antarctic pIgR genes displayed particularly long introns marked by sites of transposable elements and transcription factors. Furthermore, analysis of T. bernacchii pIgR cDNA unveiled multiple amino acid substitutions unique to the Antarctic species, all introducing adaptive features, including N-glycosylation sequons. Interestingly, C. gobio shared most features with the other perciforms rather than with the cold-adapted relatives. T. bernacchii pIgR transcripts were predominantly expressed in mucosal tissues, as indicated by q-PCR and in situ hybridization analysis. These results suggest that in cold-adapted species, pIgR preserved its fundamental role in mucosal immune defense, although remarkable gene structure modifications occurred.