Screening 5-lipoxygenase inhibitors from selected traditional Chinese medicines and isolation of the active compounds from Polygoni Cuspidati Rhizoma by an on-line bioactivity evaluation system.

To identify natural products as new prototypes for 5-lipoxygenase (5-LOX), 12 traditional Chinese medicines (TCMs) were selected for screening their 5-LOX inhibition activities. The results showed that the methanol extracts of all selected TCMs (n = 12) possessed inhibitory activities against 5-LOX at 200 µg/mL, of which six extracts of the TCMs showed significant inhibitory effects with IC50 values in the range from 33.2 ± 1.4 µg/mL to 153.5 ± 1.7 µg/mL, and the extract of Polygoni Cuspidati Rhizoma (RPC) was the most active sample. An on-line ultra-performance liquid chromatography-photodiode array-MSn -5-LOX-fluorescence detector (UPLC-PDA-MSn -5-LOX-FLD) method was applied to further identify the potential 5-LOX inhibitory constituents in RPC extracts, which resulted in the identification of seven components with 5-LOX-binding activities. Finally, four compounds (polydatin, resveratrol, emodin-8-O-glucoside, and emodin) were successfully purified from RPC extracts. The 5-LOX inhibition action was assayed in vitro, and the results showed that these compounds possessed potent inhibitory effects against 5-LOX with IC50 values of 15.3 ± 2.1, 4.5 ± 1.2, 23.8 ± 0.4, and 11.8 ± 1.5 µg/mL, respectively. This was the first study to reveal the 5-LOX inhibitory constituents of RPC, and the present investigation might provide a valuable approach for the rapid discovery of natural inhibitors from TCMs.

Cyperane-Type and Related (Nor)Sesquiterpenoids from the Root Bark of Acanthopanax gracilistylus and Their Inhibitory Effects on Nitric Oxide Production.

An enantiomeric pair of rare cyperane-type sesquiterpenoids, (+)- and (-)-gracilistones C (1a, 1b), together with a novel norsesquiterpenoid, gracilistone D (2), bearing a bicyclic lactone system were isolated from the root bark of Acanthopanax gracilistylus using LC-MS-IT-TOF analyses. The structures and absolute configurations of 1a, 1b, and 2 were elucidated by 1D and 2D NMR spectroscopy, X-ray diffraction, and ECD spectroscopic methods. Intermediate 1b suggests a possible biosynthesis process involving compound 2. The bioassay results showed that compounds 1a, 1b, and 2 exhibited significant inhibitory effects against lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells, with IC50 values of 7.7 ± 0.6, 6.8 ± 1.5, and 2.6 ± 0.4 µM, respectively. Additional docking analyses provided some perspective of this activity in human inducible nitric oxide synthase.

NO in the dPAG modulates panic-like responses and ASIC1a expression in the prefrontal cortex and hippocampus in mice.

Panic disorder (PD) is a multifactorial neuropsychiatric disorder. Our previous study has demonstrated that the nitric oxide (NO) pathway and the acid-sensing ion channel 1a (ASIC1a) level in the dorsal midbrain periaqueductal gray (dPAG) are involved in the modulation of panic-like responses. In addition, the prefrontal cortex (PFC) and the hippocampus also play a role in panic-like responses. However, no studies have investigated the protein level of ASIC1a in the PFC and hippocampus in a mouse model of panic-like disorders after alteration of the NO pathway in the dPAG. We investigated the production of a panic attack with intra-dPAG injections of SNAP, an NO donor, and 7-NI, an nNOS inhibitor. Moreover, we measured ASIC1a protein levels in the PFC and hippocampus. The rat exposure test (RET) is frequently used as an animal model of panic. In our study, C57BL/6 mice received an intra-dPAG injection of SNAP or 7-NI before RET; neurobehavioral tests were then conducted, followed by mechanistic evaluation through western blot analysis in the PFC and hippocampus. An intra-dPAG infusion of SNAP significantly increased the panic-like effect, whereas treatment with 7-NI decreased fear behavior. Mice treated with SNAP/7-NI showed significantly increased/decreased ASIC1a expression in the PFC, and a decreasing/increasing trend in the hippocampus. The present study suggests that the NO pathway in the dPAG plays a key role in panic-like responses in mice confronted by a rat, further, NO intra-dPAG injection also modulates the ASIC1a expression levels in the PFC and hippocampus.

LAMP real-time turbidity detection for fowl adenovirus.

BACKGROUND: Fowl adenovirus (FAdV) is an infectious agent that can cause jaundice, severe anaemia, dyspnoea and reproductive disorders in fowls. Since 2015, FAdV disease has been rapidly spreading among broiler chickens in China, where it has caused significant economic losses. In this study, a loop-mediated isothermal amplification (LAMP) real-time turbidity method with strong specificity to FAdV was established. RESULTS: The established assay was specific to FAdV-4, and the lower limit of detection was 75 copies/µL of extracted DNA. The positive detection rate for the suspected tissue samples was 33.3% (14/42), which was consistent with that of the real-time PCR. CONCLUSION: The proposed LAMP method can quickly and accurately detect prevalent FAdV via real-time turbidity assay, thereby providing a diagnostic platform for the prevention and control of the FAdV disease.

[Preparation and quality standard of standard decoction of Scrophulariae Radix pieces].

The standard decoction of Chinese herbal decoction pieces is a standard reference substance to measure whether different dosage forms of Chinese medicine are basically consistent with those of clinical decoction,and provides new ideas and methods for effectively solving the problems of uneven quality in Chinese medicine dispensing granules. In this study,a systematic method for evaluating the quality of Scrophulariae Radix decoction was established from the perspective of " standard decoction",providing reference for the quality control of the Scrophulariae Radix dispensing granules. 15 batches of Scrophulariae Radix decoction pieces from different origins were collected,and 15 batches of standard decoctions were prepared according to the standardized process with water as solvent.Harpagide and harpagoside were used as quantitative detection indicators to determine the content,calculate the transfer rates and determine the extraction rate. The high performance liquid chromatography( HPLC) was used to establish a standard decoction fingerprint analysis method. The results showed that the transfer rates of harpagide and harpagoside in 15 batches of Scrophulariae Radix pieces standard decoction were( 70. 84±13. 39) % and( 48. 56±6. 40) % respectively; the extraction rate was( 57. 47±5. 89) %. Nine peaks were identified in the HPLC fingerprint,and the similarity was higher than 0. 97 between the fingerprints of 15 batches of standard decoction and the control fingerprint. In this study,the preparation process of standard decoction of Scrophulariae Radix pieces conformed to the traditional decoction preparation method. The sources of the samples were representative,and the established fingerprint method was stable and feasible,which can provide reference for the preparation and quality control of Scrophulariae Radix dispensing granules.

Regulatory role of microRNA-320a in the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells by targeting estrogen-related receptor γ.

Recently, microRNAs (miRNAs) have been demonstrated to participate in many physiological and biological processes, especially by acting as circulating biomarkers or modulators in cell differentiation. Therefore, the aim of the current study was to clarify whether microRNA-320a (miR-320a) regulates the proliferation, migration, invasion, and apoptosis of trophoblasts and endothelial cells. In this study, miR-320a mimics and inhibitors were transfected into HTR.8/SVneo cells and human umbilical vein endothelial cells (HUVECs) using liposomes. Subsequently, the expression of miR-320a and estrogen-related receptor γ (ERRγ) mRNA was detected by a reverse transcription quantitative polymerase chain reaction, whereas the protein expression of ERRγ, vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1), human 3beta-hydroxysteroid dehydrogenase type 1 (HSD3B1), and human chorionic gonadotropin (HCG) was detected by western blot analysis. Furthermore, the proliferation, invasion/migration, and apoptosis of cells were analyzed by the cell counting kit-8 assay, transwell assay, and flow cytometry, respectively. The results showed that overexpression of miR-320a decreased the optical density (OD) values and the proliferation rate of HTR.8/SVneo cells and HUVECs, while inhibiting the expression of VEGF, Ang-1, HSD3B1, and HCG in these cells. Furthermore, miR-320a reduced the ability of cell invasion and migration, while increasing the rate of cell apoptosis. After cotransfecting the cells with miR-320a and ERRγ small (or short) interfering RNA (siRNA), the decreased ERRγ expression led to inhibited proliferation, migration, and invasion, but increased apoptosis of HTR.8/SVneo cells and HUVECs. Our results further revealed that miR-320a induced the apoptosis of trophoblasts and endothelial cells while inhibiting their proliferation, migration, and invasion by decreasing the expression of ERRγ and by indirectly suppressing the expression of VEGF, Ang-1, HSD3B1, and HCG.

Isolation and genetic characterization of avian influenza virus H4N6 from ducks in China.

An avian influenza virus (AIV) strain belonging to the H4 subtype and provisionally designated as A/duck/China/J1/2012(H4N6) was isolated from diseased ducks with respiratory disease at a commercial poultry farm in Shandong, China, in 2012. The genomic coding sequences of all eight segments of this J1 isolate were determined and used for subsequent analysis. Phylogenetic analysis of all eight segments showed that this duck H4N6 virus was of Eurasian lineage and not American lineage. The results show that the virus probably emerged because of a reassortment event involving other avian H4N6 and H6N1 viruses. Interestingly, this H4N6 virus had all the conserved features common to low-pathogenic AIVs, including the HA cleavage sequence, receptor-binding sequences for the 2,3-linked sialic acid receptor in avian species, and the PB2 627E motif. These results suggest that the duck H4N6 isolate could not cross the species barrier to infect and replicate in mammals, including humans. In addition, screening of the duck serum samples showed that only 0.57 % (2/352) of the individuals had weak but measurable hemagglutination inhibition (HI) antibody titers. The low antibody prevalence data were also supported by the failure to detect H4N6 virus (0/56) in clinical nasal swabs of the ducks. These data indicate an alternate reservoir for the H4N6 virus.

Genetic diversity and differentiation of the rhesus macaque (Macaca mulatta) population in western Sichuan, China, based on the second exon of the major histocompatibility complex class II DQB (MhcMamu-DQB1) alleles.

BACKGROUND: Rhesus macaques living in western Sichuan, China, have been separated into several isolated populations due to habitat fragmentation. Previous studies based on the neutral or nearly neutral markers (mitochondrial DNA or microsatellites) showed high levels of genetic diversity and moderate genetic differentiation in the Sichuan rhesus macaques. Variation at the major histocompatibility complex (MHC) loci is widely accepted as being maintained by balancing selection, even with a low level of neutral variability in some species. However, in small and isolated or bottlenecked populations, balancing selection may be overwhelmed by genetic drift. To estimate microevolutionary forces acting on the isolated rhesus macaque populations, we examined genetic variation at Mhc-DQB1 loci in 119 wild rhesus macaques from five geographically isolated populations in western Sichuan, China, and compared the levels of MHC variation and differentiation among populations with that previously observed at neutral microsatellite markers. RESULTS: 23 Mamu-DQB1 alleles were identified in 119 rhesus macaques in western Sichuan, China. These macaques exhibited relatively high levels of genetic diversity at Mamu-DQB1. The Hanyuan population presented the highest genetic variation, whereas the Heishui population was the lowest. Analysis of molecular variance (AMOVA) and pairwise FST values showed moderate genetic differentiation occurring among the five populations at the Mhc-DQB1 locus. Non-synonymous substitutions occurred at a higher frequency than synonymous substitutions in the peptide binding region. Levels of MHC variation within rhesus macaque populations are concordant with microsatellite variation. On the phylogenetic tree for the rhesus and crab-eating macaques, extensive allele or allelic lineage sharing is observed between the two species. CONCLUSIONS: Phylogenetic analyses confirm the apparent trans-species model of evolution of the Mhc-DQB1 genes in these macaques. Balancing selection plays an important role in sharing allelic lineages between species, but genetic drift may share balancing selection dominance to maintain MHC diversity. Great divergence at neutral or adaptive markers showed that moderate genetic differentiation had occurred in rhesus macaque populations in western Sichuan, China, due to the habitat fragmentation caused by long-term geographic barriers and human activity. The Heishui population should be paid more attention for its lowest level of genetic diversity and relatively great divergence from others.

mRNA Splicing of UL44 and Secretion of Alphaherpesvirinae Glycoprotein C (gC) Is Conserved among the Mardiviruses.

Marek's disease (MD), caused by gallid alphaherpesvirus 2 (GaAHV2) or Marek's disease herpesvirus (MDV), is a devastating disease in chickens characterized by the development of lymphomas throughout the body. Vaccine strains used against MD include gallid alphaherpesvirus 3 (GaAHV3), a non-oncogenic chicken alphaherpesvirus homologous to MDV, and homologous meleagrid alphaherpesvirus 1 (MeAHV1) or turkey herpesvirus (HVT). Previous work has shown most of the MDV gC produced during in vitro passage is secreted into the media of infected cells although the predicted protein contains a transmembrane domain. We formerly identified two alternatively spliced gC mRNAs that are secreted during MDV replication in vitro, termed gC104 and gC145 based on the size of the intron removed for each UL44 (gC) transcript. Since gC is conserved within the Alphaherpesvirinae subfamily, we hypothesized GaAHV3 (strain 301B/1) and HVT also secrete gC due to mRNA splicing. To address this, we collected media from 301B/1- and HVT-infected cell cultures and used Western blot analyses and determined that both 301B/1 and HVT produced secreted gC. Next, we extracted RNAs from 301B/1- and HVT-infected cell cultures and chicken feather follicle epithelial (FFE) skin cells. RT-PCR analyses confirmed one splicing variant for 301B/1 gC (gC104) and two variants for HVT gC (gC104 and gC145). Interestingly, the splicing between all three viruses was remarkably conserved. Further analysis of predicted and validated mRNA splicing donor, branch point (BP), and acceptor sites suggested single nucleotide polymorphisms (SNPs) within the 301B/1 UL44 transcript sequence resulted in no gC145 being produced. However, modification of the 301B/1 gC145 donor, BP, and acceptor sites to the MDV UL44 sequences did not result in gC145 mRNA splice variant, suggesting mRNA splicing is more complex than originally hypothesized. In all, our results show that mRNA splicing of avian herpesviruses is conserved and this information may be important in developing the next generation of MD vaccines or therapies to block transmission.

Oncogenic Animal Herpesviruses.

Oncogenic viruses play a pivotal role in oncology due to their unique role in unraveling the complexities of cancer development. Understanding the role viruses play in specific cancers is important to provide basic insights into the transformation process, which will help identify potential cellular targets for treatment. This review discusses the diverse role of animal herpesviruses in initiating and promoting various forms of cancer. We will summarize the mechanisms that underlie the development of animal herpesvirus-induced cancer that may provide a basis for developing potential therapeutic interventions or preventative strategies in the future.

Sensing combustion intermediates by femtosecond filament excitation.

Simultaneous monitoring of multiple combustion intermediates using femtosecond filament-induced nonlinear spectroscopy is demonstrated. Clean fluorescence emissions from free radicals CH, CN, NH, OH, and C(2), as well as atomic C and H, are observed when a femtosecond filament is formed in the laminar ethanol/air flame on an alcohol burner. The fluorescence signals of these species are found to vary as functions of the position of interaction of the filament with the flame along the vertical axis of the central combusting flow, opening up a possibility for remote combustion diagnostic in engines by the excitation of femtosecond laser filament.

Effect of luteinizing hormone/choriogonadotropin receptor (LHCGR) gene on chicken reproductive traits.

Luteinizing hormone/choriogonadotropin receptor (LHCGR) gene, potentially related to reproductive traits in chickens, was genotyped by using the Pooled DNA Sequencing, PCR-SSCP and Directing Sequencing techniques. 306 Erlang Mountain chickens form one line (SD03, a line that has been selected for egg quality from a local chicken breed in Sichuan province, China) were genotyped in this study. The associations between LHCGR polymorphisms and six reproductive traits [body weight at first egg (BWAFE), weight of first egg, age at first egg (AFE), number of eggs at 300 days of age (EN), body weight at 300 days of age and egg weight at 300 days of age (EWTA)] were estimated using the one-way analysis of variance method. Results showed that SNP +G4058A and SNP +T4099G of the LHCGR gene were significantly associated with BWFE and AFE. Birds with the AG genotype for the +G4058A SNP exhibited shorter AFE (P < 0.05) and greater EN than those of the GG and AA genotypes, suggesting a balancing selection (overdominance); the effect of allele C in SNP +C3021T and allele C in SNP +T4490C on EN and AFE is additive and may reflect the influence of positive selection. These alleles have promise as genetic markers for future marker-assisted selection.

Genetic variation and phylogenetic relationship between three serow species of the genus Capricornis based on the complete mitochondrial DNA control region sequences.

The molecular evidence of phylogenetic status regarding the Formosan serow (Capricornis swinhoei) is not robust and little is known about the genetic diversity of the Sumatran serow (Capricornis sumatraensis), which partly is due to the hardness in sample collection. Here we determined the sequences of the complete mitochondrial DNA control region (1,014 bp) of 19 Sumatran-serow individuals. Nine new haplotypes were defined based on 78 variable sites. Combined analysis with other 32 haplotypes downloaded from the public database, including 1 Sumatran-serow, 11 Formosan-serow and 20 Japanese-serow (Capricornis crispus) haplotypes, a relatively high level of nucleotide diversity was first observed in Sumatran serow (π = 0.0249). By comparative analysis with structural consensus sequences from other mammals, we have identified central, left and right domains and depicted the putative functional structure, including extend termination associated sequences and conserve sequence blocks, in mtDNA control region. The alignment of mtDNA control region revealed that both Sumatran and Japanese serow have two tandem repeats (TRs), but three TRs in Formosan serow. Phylogenetic analyses revealed that the Formosan serow is distinct species with the Japanese serow, but a sister group with the Sumatran serow. The divergence time estimated among three serow species revealed that Pleistocene climate changes and the uplift of Qinghai-Tibetan plateau might play an important role in the genetic differentiation of the serows. These results mainly provide the convinced evidence on the genetic relationship between three serow species.

Genetic diversity and population structure in wild Sichuan rhesus macaques.

Because wild rhesus macaque (Macaca mulatta) populations have suffered major declines, there is a growing need to characterize their genetic and population structure in order to protect the genetic integrity of this species. In this study, we genotyped a sample comprising 120 wild rhesus macaques from six sites in Sichuan Province for 30 nuclear microsatellite (STR) loci using an ABI 3130xl genetic analyzer. Bayesian analyses and PCA clearly differentiated monkeys from Heishui from those at other sites. The samples from all six sites exhibited high gene diversity suggesting that the Sichuan wild rhesus macaque populations are not threatened by a lack of genetic diversity. Deviation from Hardy-Weinberg equilibrium was more frequent in the Danba and Heishui populations. This may be due to the more fragmented habitat and less disturbance by humans in this area that foster greater subpopulation structuring than occurs in eastern China. We suggest that this population subdivision is the result of both long-term geographic barriers and human activity.

Autophagy modulation as a target for anticancer drug discovery.

Autophagy, an evolutionarily conserved catabolic process involving the engulfment and degradation of non-essential or abnormal cellular organelles and proteins, is crucial for homeostatic maintenance in living cells. This highly regulated, multi-step process has been implicated in diverse diseases including cancer. Autophagy can function as either a promoter or a suppressor of cancer, which makes it a promising and challenging therapeutic target. Herein, we overview the regulatory mechanisms and dual roles of autophagy in cancer. We also describe some of the representative agents that exert their anticancer effects by regulating autophagy. Additionally, some emerging strategies aimed at modulating autophagy are discussed as having the potential for future anticancer drug discovery. In summary, these findings will provide valuable information to better utilize autophagy in the future development of anticancer therapeutics that meet clinical requirements.

Melatonin receptor genes in vertebrates.

Melatonin receptors are members of the G protein-coupled receptor (GPCR) family. Three genes for melatonin receptors have been cloned. The MT1 (or Mel1a or MTNR1A) and MT2 (or Mel1b or MTNR1B) receptor subtypes are present in humans and other mammals, while an additional melatonin receptor subtype, Mel1c (or MTNR1C), has been identified in fish, amphibians and birds. Another melatonin related orphan receptor, GPR50, which does not bind melatonin, is found exclusively in mammals. The hormone melatonin is secreted primarily by the pineal gland, with highest levels occurring during the dark period of a circadian cycle. This hormone acts systemically in numerous organs. In the brain, it is involved in the regulation of various neural and endocrine processes, and it readjusts the circadian pacemaker, the suprachiasmatic nucleus. This article reviews recent studies of gene organization, expression, evolution and mutations of melatonin receptor genes of vertebrates. Gene polymorphisms reveal that numerous mutations are associated with diseases and disorders. The phylogenetic analysis of receptor genes indicates that GPR50 is an outgroup to all other melatonin receptor sequences. GPR50 may have separated from a melatonin receptor ancestor before the split between MTNR1C and the MTNR1A/B ancestor.

Monoterpenoids from the root bark of Acanthopanax gracilistylus and their inhibitory effects on neutrophil elastase, 5-lipoxygenase, andcyclooxygenase-2 in vitro.

Twenty-four monoterpenoids, including three previously undescribed compounds (1-3), were isolated from the root bark of Acanthopanax gracilistylus W. W. Smith (Acanthopanacis Cortex). Their structures were unambiguously established based on spectroscopic analysis (HR-ESIMS, IR, 1D, and 2D NMR), and the absolute configurations of 1-3 were elucidated by comparing their experimental and calculated electronic circular dichroism spectra. In addition, the structure of 8 was confirmed by single-crystal X-ray diffraction. The inhibitory activities of 1-24 against neutrophil elastase, 5-lipoxygenase, and cyclooxygenase-2 (COX-2) were studied in vitro for the first time, and the results showed that compound 24 possessed a significant inhibitory effect on COX-2 with an IC50 value of 1.53 ± 0.10 µΜ. This research first reported the presence of monoterpenoids in Acanthopanacis Cortex, including one monoterpenoid 2 with an unusual 4/5 bicyclic lactone system, and compounds 4 and 5 have never been reported in nature.

Resonant gain suppression and superluminal group velocity in a multilevel system.

We investigate the interaction of an open (N + 1)-level extended V-type atomic system (i.e. a closed (N + 2)-level atomic system) with N coherent laser fields and one incoherent pumping field through both analytical and numerical calculations. Our results show that the system can exhibit multiple resonant gain suppressions via perfect quantum destructive interference, which is usually believed to be absent in closed three-level V system and its extended versions involving more atomic levels, with at most N - 1 transparency windows associated with very steep anomalous dispersions occurring in the system. The superluminal group velocity of the probe-laser pulse with at most N - 1 negative values can also be generated and controlled with little gain or absorption.

[Polymorphic analysis of Mhc-DPB1 gene exon 2 in Tibetan macaques (Macaca thibetana)].

Major histocompatibility complex (MHC) molecules play an important role in the susceptibility and/or resistance to many diseases. To gain an insight into the MHC background of the Tibetan macaques (Macaca thibetana), and thereby facilitate their protection and application in biomedical research, the second exon of the Mhc-DPB1 genes from 70 Tibetan macaques in Sichuan Province were characterized by PCR, cloning, sequencing, and statistical analysis. A total of 18 Mhc-DPB1 alleles were identified from Tibetan macaques, of which one (Math-DPB1*01:06N) was a pseudogene. Math-DPB1*06:01:01 (67.14%) was the most frequent allele in all the 18 alleles detected, followed by Math-DPB1* 01:03:01 (37.14%), Math-DPB1*09:02 (25.71%), and Math-DPB1*22:01 (15.71%). The alignment of putative amino acid sequences of the 18 Math-DPB1 alleles showed that 5 variable sites were species-specific to Tibetan macaques. A phylogenetic tree constructed using DPB1 alleles in difference species demonstrated that the alleles for Math-DPB1, Mamu-DPB1, and Mafa-DPB1 tended to mix together, rather than cluster into a separate branch in a species-specific fashion, and the Trans-species polymorphism was also observed in the phylogenetic tree. Selection analysis revealed that balancing selection may play an important role in maintaining the polymorphism of Math-DPB1 genes.

The Conserved Herpesviridae Protein Kinase (CHPK) of Gallid alphaherpesvirus 3 (GaHV3) Is Required for Horizontal Spread and Natural Infection in Chickens.

We have formerly identified the conserved herpesvirus protein kinase (CHPK) as essential for horizontal transmission of Marek's disease virus (MDV). Thus far, it has been confirmed that the mutation of the invariant lysine (K) of CHPKs abrogates kinase activity and that CHPK activity is required for MDV horizontal transmission. Since CHPK is conserved among all members of the Herpesviridae, we hypothesized that CHPK, and specifically its kinase activity, is important for the horizontal transmission of other herpesviruses. To test this hypothesis, we utilized our experimental and natural infection model in chickens with MD vaccine strain 301B/1 of Gallid alphaherpesvirus 3 (GaHV3). First, we mutated the invariant lysine (K) 157 of 301B/1 CHPK to alanine (A) and determined whether it was required for horizontal transmission. To confirm the requirement of 301B/1 CHPK activity for transmission, a rescued virus was generated in which the A157 was changed back to a K (A157K). Despite both the CHPK mutant (K157A) and rescuant (A157K) viruses having replication defects in vivo, only the CHPK mutant (K157A) was unable to spread to contact chickens, while both wild-type and rescuant (A157K) viruses transmitted efficiently, confirming the importance of CHPK activity for horizontal spread. The data confirm that CHPK is required for GaHV3 transmission and suggest that the requirement of avian CHPKs for natural infection is conserved.