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Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex

The ongoing SARS (severe acute respiratory syndrome)-CoV (coronavirus)-2 pandemic has exposed major gaps in our knowledge on the origin, ecology, evolution, and spread of animal coronaviruses. Porcine epidemic diarrhea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae that may have originated from bats and leads to significant hazards and widespread epidemics in the swine population. The role of local and global trade of live swine and swine-related products in disseminating PEDV remains unclear, especially in developing countries with complex swine production systems. Here, we undertake an in-depth phylogeographic analysis of PEDV sequence data (including 247 newly sequenced samples) and employ an extension of this inference framework that enables formally testing the contribution of a range of predictor variables to the geographic spread of PEDV. Within China, the provinces of Guangdong and Henan were identified as primary hubs for the spread of PEDV, for which we estimate live swine trade to play a very important role. On a global scale, the United States and China maintain the highest number of PEDV lineages. We estimate that, after an initial introduction out of China, the United States acted as an important source of PEDV introductions into Japan, Korea, China, and Mexico. Live swine trade also explains the dispersal of PEDV on a global scale. Given the increasingly global trade of live swine, our findings have important implications for designing prevention and containment measures to combat a wide range of livestock coronaviruses. ispartof: MOLECULAR BIOLOGY AND EVOLUTION vol:39 issue:2 ispartof: location:United States status: published

Emerging infectious diseases, especially if caused by bat-borne viruses, significantly affect public health and the global economy. There is an urgent need to understand the mechanism of interspecies transmission, particularly to humans. Viral genetics; host factors, including polymorphisms in the receptors; and ecological, environmental, and population dynamics are major parameters to consider. Here, we describe the taxonomy, geographic distribution, and unique traits of bats associated with their importance as virus reservoirs. Then, we summarize the origin, intermediate hosts, and the current understanding of interspecies transmission of Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, Nipah, Hendra, Ebola, Marburg virus, and rotaviruses. Finally, the molecular interactions of viral surface proteins with host cell receptors are examined, and a comparison of these interactions in humans, intermediate hosts, and bats is conducted. This uncovers adaptive mutations in virus spike protein that facilitate cross-species transmission and risk factors associated with the emergence of novel viruses from bats.

Porcine viral diarrhea diseases affect the swine industry, resulting in significant economic losses. Porcine epidemic diarrhea virus (PEDV) genotypes G1 and G2, and groups A and C of the porcine rotavirus, are major etiological agents of severe gastroenteritis and profuse diarrhea, particularly among piglets, with mortality rates of up to 100%. Based on the high prevalence rate and frequent co-infection of PEDV, RVA, and RVC, close monitoring is necessary to avoid greater economic losses. We have developed a multiplex TaqMan probe-based real-time PCR for the rapid simultaneous detection and differentiation of PEDV subtypes G1 and G2, RVA, and RVC. This test is highly sensitive, as the detection limits were 20 and 100 copies/μL for the G1 and G2 subtypes of PEDV, respectively, and 50 copies/μL for RVA and RVC, respectively. Eighty-eight swine clinical samples were used to evaluate this new test. The results were 100% in concordance with the standard methods. Since reassortment between porcine and human rotaviruses has been reported, this multiplex test not only provides a basis for the management of swine diarrheal viruses, but also has the potential to impact public health as well.

Schwann cells (SCs) are PNS glia that play numerous support functions including myelination of axons. After PNS injury, SCs facilitate regeneration by phagocytosing cellular debris and providing physical and biochemical cues to guide axon growth. This reparative phenotype suggests SCs could be critical cellular targets for enhancing nerve regeneration. One method for altering cell morphology and motility is the application of direct current (DC) electric fields (EFs). Endogenous EFs have physiologic relevance during embryogenesis and serve as guidance and polarization cues. While much literature exists on EFs and CNS and PNS neurons, the effects of EFs on SCs have not been extensively studied. In this work, cell alignment, migration, and morphology of rat SCs were measured in response to several EF stimulation regimes including constant DC, 50% duty cycle DC and oscillating DC. SCs were found to re-orient perpendicular to field lines and respond to DC EFs as low as 75 mV/mm. EF exposure promoted directed migration, with travel towards the cathode at a mean rate of 7.5 µm/h. The data highlight the utility of EFs in modulating SC morphology, alignment and migration. Results may have implications for using EFs to attract and realign SCs at the site of PNS trauma.

The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.

Pair correlation functions provide a summary statistic which quantifies the amount of spatial correlation between objects in a spatial domain. While pair correlation functions are commonly used to quantify continuous-space point processes, the on-lattice discrete case is less studied. Recent work has brought attention to the discrete case, wherein on-lattice pair correlation functions are formed by normalizing empirical pair distances against the probability distribution of random pair distances in a lattice with Manhattan and Chebyshev metrics. These distance distributions are typically derived on an ad hoc basis as required for specific applications. Here we present a generalized approach to deriving the probability distributions of pair distances in a lattice with discrete Manhattan and Chebyshev metrics, extending the Manhattan and Chebyshev pair correlation functions to lattices in k dimensions. We also quantify the variability of the Manhattan and Chebyshev pair correlation functions, which is important to understanding the reliability and confidence of the statistic.

Cats are susceptible to a wide range of influenza A viruses (IAV). Furthermore, cats can serve as an intermediate host, and transfer avian influenza virus (AIV) H7N2 to a veterinarian. In this report, a novel reassortant influenza virus, designated A/feline/Jiangsu/HWT/2017 (H3N2), and abbreviated as FIV-HWT-2017, was isolated from nasal swab of a symptomatic cat in Jiangsu province, China. Sequence analysis indicated that, whilst the other seven genes were most similar to the avian-origin canine influenza viruses (CIV H3N2) isolated in China, the NS gene was more closely related to the circulating human influenza virus (H3N2) in the region. Therefore, FIV-HWT-2017 is a reassortant virus. In addition, some mutations were identified, and they were similar to a distinctive CIV H3N2 clade. Whether these cats were infected with the reassortant virus was unknown, however, this random isolation of a reassortant virus indicated that domestic or stray cats were “mixing vessel” for IAV cannot be ruled out. An enhanced surveillance for novel influenza virus should include pet and stray cats.

Prior to the emergence of H3N8 canine influenza virus (CIV) and the latest avian-origin H3N2 CIV, there was no evidence of a circulating canine-specific influenza virus. Molecular and epidemiological evidence suggest that H3N8 CIV emerged from H3N8 equine influenza virus (EIV). This host-range shift of EIV from equine to canine hosts and its subsequent establishment as an enzootic CIV is unique because this host-range shift was from one mammalian host to another. To further understand this host-range shift, we conducted a comprehensive phylodynamic analysis using all the available whole-genome sequences of H3N8 CIV. We found that (1) the emergence of H3N8 CIV from H3N8 EIV occurred in approximately 2002; (2) this interspecies transmission was by a reassortant virus of the circulating Florida-1 clade H3N8 EIV; (3) once in the canine species, H3N8 CIV spread efficiently and remained an enzootic virus; (4) H3N8 CIV evolved and diverged into multiple clades or sublineages, with intra and inter-lineage reassortment. Our results provide a framework to understand the molecular basis of host-range shifts of influenza viruses and that dogs are potential “mixing vessels” for the establishment of novel influenza viruses.

Growth in biological systems occurs as a consequence of cell proliferation fueled by a nutrient supply. In general, the nutrient gradient of the system will be nonconstant, resulting in biased cell proliferation. We develop a uniaxial discrete cellular automaton with biased cell proliferation using a probability distribution which reflects the nutrient gradient of the system. An explicit probability mass function for the displacement of any tracked cell under the cellular automaton model is derived and verified against averaged simulation results; this displacement distribution has applications in predicting cell trajectories and evolution of expected site occupancies.

H3N2 canine influenza virus (CIV) originated from avian species and emerged in dogs in Asia around 2005 where it became enzootic before reaching the USA in 2015. To investigate the key aspects of the evolution of H3N2 CIV regarding its emergence and adaptation in the canine host, we conducted an extensive analysis of all publicly available H3N2 CIV sequences spanning a 10-year period. We believe that H3N2 AIVs transferred to canines around 2002-2004. Furthermore, H3N2 CIVs could be divided into seven major clades with strong geographic clustering and some changed sites evidence of adaptive evolution. Most notably, the dN/dS of each H3N2 CIVs segment was higher than the correspondent of H3N2 AIVs and the U content of HA and NA was increasing over time, suggesting the idea that this avian-origin virus may be gradually adapting to the host. Our results provide a framework to elucidate a general mechanism for emergence of novel influenza viruses.

Avian influenza A(H7N9) virus and mixed live poultry–animal markets in Guangdong province: a perfect storm in the making?

Mitogenic activity of UV-inactivated influenza viruses in cord blood lymphocytes (CBL), as measured by cytokine release, was investigated. Using prototype viruses of subtype H3N2 (A/Aichi/68), H2N2 (A/Japan/57), and H1N1 (A/Puerto Rico/34) for influenza A virus, and B/Lee/40 for influenza B virus, the results indicated that both Th1 and Th2 cytokines were induced. Stimulation indices were significantly higher for IFNγ, IL-4 and IL-10 by influenza A viruses than by influenza B virus. Stimulation indices for IL-2 and IL-6 were lower, as these two cytokines were spontaneously released by cord blood lymphocytes in culture. Alignment of the amino acid sequences of the HA for the viruses used in this study indicated that influenza B virus lacked sequence homology to the antigenic sites identified for influenza A virus. Therefore, the antigenic sites may play a role in the mitogenic property, and cord blood lymphocytes could provide a system to compare this property for clinical isolates of influenza virus.

Phylogenetic and antigenic analyses indicate that recent circulating equine-2 influenza viruses in the United States have been alternating between two genetic and antigenic distinct lineages since 1996. The evolution rates for these two lineages, the Kentucky and the Florida lineage, are very similar. For the earlier isolates in the Kentucky lineage, there are multiple and sequential nonsynonymous substitutions at antigenic sites B and D. However, there are no changes at any of these antigenic sites for KY98 and OK00. In the Florida lineage, except for NY99 with one amino acid substitution at antigenic site B, viruses in this lineage do not have nonsynonymous substitutions at any of the antigenic sites. The lack of amino acid substitutions at these antigenic sites suggests a mechanism other than immune selection is responsible for the maintenance of these viral lineages. Serological analysis indicates that these two lineages are antigenic distinct, and the pattern of reactivity of horse sera towards these two lineages alternates in consecutive years, parallel to the "switching" of virus lineage seen in the phylogenetic tree. This alternate circulation may play a role in the maintenance of these two lineages of equine-2 influenza virus.

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.

Zoonotic transmission of pathogenic avian influenza virus (AIV) is a potential public health threat ([1][1], [2][2]), as the virus may acquire human-to-human transmissibility through mutations or by reassortment with seasonal influenza viruses (e.g., H3N2 and H1N1). The recent outbreak of novel H7N9

The expression of hepatitis B virus (HBV) DNA in the liver was studied by nonisotopic in situ hybridization and correlated with liver histology, different phases in the natural evolution of chronic hepatitis B, and hepatic expression of HBV antigens in 251 Chinese patients with chronic HBV infection. A good correlation was found between the detection of HBV-DNA by in situ hybridization and serum HBV-DNA (P < .01). Chronic active hepatitis had the highest HBV-DNA detected in cytoplasm and nuclei, compared with livers showing minimal change, chronic persistent hepatitis, cirrhosis, and hepatocellular carcinoma. HBV-DNA in cytoplasm exceeded HBV-DNA in nucleus in all patients except in livers with hepatocellular carcinoma. Hepatic HBV-DNA correlated with disease activity (P < .02) and the correlation was highly significant with intralobular activity (P < .001). Patients in the early viral replicative phase of infection had higher levels of cytoplasmic and nuclear HBV-DNA compared with the late viral nonreplicative phase. Cytoplasmic and nuclear HBV-DNA correlated with hepatic expression of HBcAg and HBsAg (P < .05 in both cases), but not with HBeAg. These data indicate that hepatic expression of HBV-DNA follows the natural history of chronic HBV infection and is associated with active liver disease.

Persistent viral infections in vitro are useful systems to study the coevolution of virus and cell populations. Persistent infection of mouse Friend erythroleukemic cells (FEL) with vaccinia virus results in profound changes of the virus as well as of the cells. To investigate phenotypic changes of other cell types, we have established a persistent infection with vaccinia virus in a human leukemic cell line (K562). This cell line can be induced to differentiate along the erythroid pathway synthesizing embryonic and fetal globins, thus providing a system in which specific genes can be stimulated. After serial passage, the persistently infected cells (K562vac) became spontaneously differentiated, as shown by the increase in the number of cells producing hemoglobin (benzidine positive cells), and resistant to superinfection. These phenotypic changes of the cells were not accompanied by changes in the viral population. Hybridization of cellular RNA with cloned embryonic and fetal globin genes indicated that uninduced K562 cells do not express these genes, whereas cells induced by hemin or butyrate express G gamma (fetal globin) epsilon and zeta (embryonic globins) genes. By contrast vaccinia infected cells spontaneously express the G gamma gene. These results demonstrate that persistent infection with vaccinia virus elicited phenotypic changes in the infected cell population; in this case the constitutive expression of fetal hemoglobin.

Persistent viral infections in vitro are useful to study the evolution of virus populations in the absence of immunological pressure. Several deletion mutants have been isolated in this laboratory from Friend erythroleukemia cells persistently infected with vaccinia virus strain IHD-W, designated SQA vac . Two of the mutants, which remain stable after serial passage in L cells, have been characterized. The deletion which range between 20 to 22 kb, has been localized at the left terminus comprising Hin dIII fragments C and N. In addition, H in dIII B fragment lost the sequences that hybridize to pAG5, a plasmid containing the 3.5 kb terminal sequences and acquired different restriction sites. Phenotypic characterization of these mutants revealed that they were not replication defective since they grew in all cell lines tested and produced plaques of the same size as the wild-type. However, they were less effective in suppressing host protein synthesis. The LD 50 for the mutants titered in NIH Swiss female mice was greater than 10 9 PFU as compared to 10 6 PFU for the wild-type, indicating reduced virulence in vivo. These mutants, which display different properties to previously described mutants with deletions at the left terminus, provide another valuable system to study the molecular basis of virulence of vaccinia.

Understanding the molecular basis of virulence in poxvirus is of great importance for the development of recombinant vaccines using vaccinia virus as a vector. We have previously described mutants of vaccinia virus with deletions ranging from 20 to 21 kb at the left terminus and with attenuated phenotype. The virulence of these mutants was studied, using different routes of inoculation, for protection from wild-type challenge in mice and for replication in vivo. Regardless of the route of inoculation, the LD50 of the deletion mutants is at least 1000-fold higher than that of the wild-type. Results from protection experiments using viable and ultraviolet-inactivated viruses, and from determination of infectivity in different organs, indicated that the mutants were unable to replicate in vivo. Southern blot hybridization of viral DNA with pSC16, a plasmid containing the vaccinia growth factor (VGF) gene, revealed that in the IHD-W strain of vaccinia virus this gene is localized at the left terminus exclusively and that the mutants lack this gene. The results suggest that absence of the VGF gene is correlated with inability to replicate in vivo and decreased virulence.