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The diagnosis of a concurrent infection of Avibacterium paragallinarum and fowl adenovirus (FAdV) in an infectious coryza-like outbreak in the outskirt of Beijing is reported. The primary signs of the infection were acute respiratory signs, a drop in egg production, and the presence of hydropericardium-hepatitis syndrome-like gross lesions. Laboratory examination confirmed the presence of A. paragallinarum by bacterial isolation and a species-specific PCR test. In addition, conventional serotyping identified the isolates as Page serovar A. Fowl adenovirus was isolated from chicken liver specimen and identified by hexon gene amplification. In addition, histopathologic analysis and transmission electron microscopy examination further confirmed the presence of the virus. Both hexon gene sequencing and phylogenetic analysis defined the viral isolate as FAdV-4. The pathogenic role of A. paragallinarum and FAdV was evaluated by experimental infection of specific-pathogen-free chickens. The challenge trial showed that combined A. paragallinarum and FAdV infection resulted in more severe clinical signs than that by FAdV infection alone. The concurrent infection caused 50% mortality compared with 40% mortality by FAdV infection alone and zero mortality by A. paragallinarum infection alone. To our knowledge, this is the first report of A. paragallinarum coinfection with FAdV. The case implies that concurrent infections with these 2 agents do occur and more attention should be given to the potential of multiple agents during disease diagnosis and treatment.

MotivationAmplicon-based nanopore sequencing is increasingly used for molecular surveillance during epidemics (e.g. ZIKA, EBOLA) or pandemics (e.g. SARS-CoV-2). However, there is still a lack of versatile and easy-to-use tools that allow users with minimal bioinformatics skills to perform the main steps of downstream analysis, from quality testing to SNPs effect to phylogenetic analysis.ResultsHere, we present ONTdeCIPHER, an amplicon-based Oxford Nanopore Technology (ONT) sequencing pipeline to analyze the genetic diversity of SARS-CoV-2 and other pathogenes. Our pipeline integrates 13 bioinformatics tools. With a single command line and a simple configuration file, users can pre-process their data and obtain the sequencing statistics, reconstruct the consensus genome, identify variants and their effects for each viral isolate, infer lineage and, finally perform multi-sequence alignments and phylogenetic analyses.Availability and implementation:ONTdeCIPHER is available at https://github.com/emiracherif/ONTdeCIPHERContactemira.cherif@ird.frSupplementary informationSupplementary data are available at …

We report a nearly complete genome sequence of Ndumu virus (NDUV) identified using a metagenomics approach. The sequence was derived from a viral isolate obtained from a bovine calf following a diagnostic investigation of the 1997 to 1998 Rift Valley fever (RVF) outbreak in the Garissa District of northeastern Kenya.

SummaryIn this study we profiled vaccine-induced polyclonal antibodies as well as plasmablast derived mAbs from individuals who received SARS-CoV-2 spike mRNA vaccine. Polyclonal antibody responses in vaccinees were robust and comparable to or exceeded those seen after natural infection. However, the ratio of binding to neutralizing antibodies after vaccination was greater than that after natural infection and, at the monoclonal level, we found that the majority of vaccine-induced antibodies did not have neutralizing activity. We also found a co-dominance of mAbs targeting the NTD and RBD of SARS-CoV-2 spike and an original antigenic-sin like backboost to seasonal human coronaviruses OC43 and HKU1. Neutralizing activity of NTD mAbs but not RBD mAbs against a clinical viral isolate carrying E484K as well as extensive changes in the NTD was abolished, suggesting that a proportion of vaccine induced RBD binding antibodies may provide substantial protection against viral variants carrying single E484K RBD mutations.

SARS-CoV-2 isolates from long-term COVID-19 patients play a significant role in understanding the mechanisms of infection and virus persistence. This study describes a SARS-CoV-2 isolate from a 53-year-old woman from Apulia (Italy), who was COVID-19 positive for approximately four months. In this paper we aimed to investigate any potential correlation between genetic mutations and clinical features of this case of infection. The viral isolate was assigned to lineage B.1.177.51 through whole-genome sequencing (WGS) and harbored a novel set of mutations on the Spike protein (V143D, del144/145 and E484K), furthermore, seroneutralization assays showed impaired response of the surveyed strain to BNT162b2 (Comirnaty) Pfizer/BioNTech vaccine-induced (average reduction of 70%) and convalescent sera (average reduction of 19.04%), when compared to VOC P.1. This study highlights the importance of genomic surveillance for the management of the COVID-19 pandemic, the relevance of monitoring of emerging SARS-CoV-2 mutations in all lineages, and the necessity of testing the response of emerging variants to available therapies and vaccines.

Zika virus (ZIKV) has spread in the Americas since 2015 and the potential establishment of a sylvatic transmission cycle in the continent has been hypothesized. We evaluated vector competence of five sylvatic Neotropical mosquito species to two ZIKV isolates. Distinct batches of Haemagogus leucoceleanus, Sabethes albiprivus, Sabethes identicus, Aedes terrens and Aedes scapularis females were respectively orally challenged and inoculated intrathoracically with ZIKV. Orally challenged mosquitoes were refractory or exhibited low infection rates. Viral dissemination was detected only in Hg. leucocelaenus, but with very low rates. Virus was not detected in saliva of any mosquito orally challenged with ZIKV, regardless of viral isolate and incubation time. When intrathoracically injected, ZIKV disseminated in high rates in Hg. leucocelaenus, Sa. identicus and Sa. albpiprivus, but low transmission was detected in these species; very low dissemination and no transmission was detected in Ae. terrens and Ae. scapularis. Together these results suggest that genetically determined tissue barriers, especially in the midgut, play a vital role in inhibiting ZIKV for transmission in the tested sylvatic mosquito species. Thus, an independent enzootic transmission cycle for ZIKV in South America is very unlikely.

This review is intended to discuss published work focused on the immunization of swine against porcine reproductive and respiratory syndrome virus (PRRSV). Many different strategies have been pursued by researchers in the quest for control and ultimately the eradication of porcine reproductive and respiratory syndrome (PRRS). Some of the strategies have lead to the licensure of commercial products while others have provided insight into the biology of PRRSV. Many of the strategies examined here have contributed to the overall understanding of the host’s immune response to PRRSV. Some strategies have provided unconventional or in some cases controversial methods of protecting swine from PRRS. The main area of interest in this review will be the clinical protection of swine from virulent PRRSV challenge. Gross lung pathology and viremia along with virus neutralizing antibody titers will serve as the primary parameters for protection in the respiratory challenge model, whereas the number of live born pigs and the number of weaned pigs along with piglet viremia will serve as the primary parameters for protection in the reproductive challenge model. Despite the incomplete reporting of these primary parameters, some publications will be discussed that have focused on protection against PRRSV by either evaluating the immune responses in non-target animals or studies conducted in swine, some without a virulent challenge. It is important to note that for the purposes of this review a homologous PRRSV challenge will be defined as a challenge with the same viral isolate from which the

a b s t r a c t Cyprinid herpesvirus-3 (CyHV-3) induces the highly contagious koi herpesvirus disease (KHVD) and may result in significant economic losses to the ornamental and food-producing carp industry. Suspicion of KHVD is triggered by clinical signs and confirmed using laboratory techniques. The latter are labour- and time-consuming, require specialised equipment and trained personnel. For rapid, on-site detection of CyHV-3, a lateral flow device (LFD) was developed using two monoclonal antibodies directed towards the viral glycoprotein ORF65. The LFD was highly specific with analytical and diagnostic specificities of 100%. Analytical sensitivity ranged between 1.25 × 102 and 2.40 × 104 plaque forming units per ml for isolates originating from geographically distinct regions. In experimentally infected carp, CyHV-3 was detected as early as 4-5 days post infection. Diagnostic sensitivities of 52.6% and 72.2% relative to PCR were recorded, depending on the viral isolate used. When onset of mortality was taken as reference, diagnostic sensitivities increased to 67.0% and 93.3%. The diagnostic sensitivity for freshly found-dead animals was 100%, irrespective of the virus isolate used. Given the high specificity and ease-of-use for on-site detection of CyHV-3, the LFD was regarded fit for purpose as a first-line diagnostic tool for the identification of acute CyHV-3 infections in KHVD affected (koi) carp.

Mammalian reoviruses exhibit a propensity to replicate in transformed cells. It is currently believed that the interferon-inducible RNA-dependent protein kinase (PKR), an intracellular host-cell resistance factor that is inhibited by an activatedRas-dependent pathway in transformed cells, is responsible for this discrimination. In the present study, reovirus isolates differing in their sensitivity to interferon were obtained by chemical mutagenesis, and examined for their replicative properties in parental andRas-transformed mouse NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i., and that there is a correlation between the ability to discriminate between transformed and parental cells, and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent onRasactivation than any other viral isolate. Altogether, this suggests that optimal reovirus isolates could be selected to attack tumour cells depending on the nature of the alterations in interferon-inducible pathways found in these cells.

Inhibiting the HIV-1 entry process offer a new therapeutic target and the hope to potentialize our current treatments against wild-type or drug-resistant viruses. Several inhibitors of CD4, co-receptor CCR5 or CXCR4 and fusion are at various levels of clinical development. How best to use this class of drugs in our therapeutic arsenal remains to be defined. It is likely that these compounds will not be used as monotherapy. Therefore, it is important to evaluate how these drugs will interact among themselves as well as with antiretrovirals from other classes. Drug interactions can range from synergy to antagonism depending on factors including binding affinity, drug concentrations, and pharmacokinetics. In the case of entry inhibitors, one must also consider that the entry of HIV-1 into the cell is a multi-step process that involve cumulative events which are interdependent. Furthermore, polymorphism both in the coreceptors and in gp120, the density of coreceptors, and the binding site of the drug may also affect efficacy. Therefore it is difficult to predict how blocking one step of the process will affect the subsequent one without carefully studying interactions of each potential combination in an in vitro system. So far, studies of interactions between fusion inhibitors and coreceptor inhibitors have shown a high level of synergy. Similar studies performed with two co-receptor inhibitors have shown results varying from synergy to high antagonism depending on the viral isolate and the compounds used. In the following chapter, we will review some concepts of mechanisms that may affect these interactions.

Phenotypic and genotypic resistance assays-methods to measure the susceptibility of HIV to drug therapy-are becoming widely available to clinicians for use in aiding long-term antiretroviral treatment planning. Phenotypic tests assess the actual response of a patient's viral isolate to individual antiretroviral drugs in culture, and genotypic tests sequence viral DNA, providing an inferred measure of resistance based on the evaluator's knowledge of established HIV-1 genetic mutational patterns for resistance to antiretroviral drugs. These tests, when used as intended, can provide information useful in planning successful antiretroviral regimens. Both types of assays have been shown to provide reliable and reproducible measures of resistance, with certain caveats: accuracy depends on the experience of the interpreter and laboratory; results from the available tests are not interchangeable, and clinically relevant thresholds of resistance have not been fully defined. Because of pharmacokinetic and cross-resistance issues, the extrapolation of single-drug data to combination regimens is difficult to accomplish and must be pooled with treatment and viral load history to best identify effective subsequent treatment regimens. This article describes the experience to date establishing the efficacy, accuracy, reproducibility, and limitations of the phenotypic and genotypic resistance assays used today.

During the twentieth century, the introduction of vaccines changed the history of mankind, eliminating most of the childhood diseases that used to cause millions of deaths. However, where conventional vaccinology approaches failed, many new and emerging infectious diseases remain a threat to health worldwide. The advent of whole-genome sequencing and innovation in bioinformatic tools radically changed the way to design and develop new and improved vaccines, starting from the genomic information of a single bacterial or viral isolate, with a process named reverse vaccinology. As the genomic era progressed, reverse vaccinology has evolved in combination with different approaches, such as transcriptomics, metabolomics, structural genomics, proteomics, and immunomics, contributing to the design of new and universal vaccines. Furthermore, the genomic information of the host is increasingly being used to aid understanding of the human immune response to vaccines.

The effect of 3 drug combinations (AZT/FLT, AZT/ddI and FLT/ddI) upon the replication of AZT-sensitive and -resistant human immuno-deficiency virus (HIV) was studied. The 3 combinations synergistically inhibited drug sensitive virus. However, AZT resistant virus showed an altered response to the combinations containing AZT: synergy was replaced by addition or antagonism. Thus, the susceptibility to a drug may affect the synergistic effect of combinations containing that drug. Other drug combinations may not be affected, since the AZT resistant virus retained a synergistic response to the combination of FLT/ddI. The synergistic effect could be regained upon reversion of resistance; a viral isolate taken after cessation of therapy, which had reverted to sensitivity to AZT, regained the synergistic response to drug combinations containing AZT. These results have implications for the use of combination chemotherapy to treat infection with HIV. Drug combinations will be most useful if the virus is sensitive to all components in the combination. It might be preferable to avoid the inclusion in combinations of drugs to which there is resistance, since we saw an antagonistic effect for combinations containing AZT in AZT resistant virus. Other combinations not containing the drug to which resistance has arisen may maintain their synergistic effect and remain good choices. Thus, a strategy of monitoring drug sensitivity and altering the combination therapy accordingly would appear to offer promise for the treatment of HIV infection.

We report the identification of an influenza A(H1N1) viral isolate found to be resistant to oseltamivir. This is the first report of resistance of H1N1 to this medication in the United Arab Emirates. Sequencing of the isolate showed that it has the H275Y mutation.

In autumn 2008, several captive red deer in the North of France were found to be serologically positive for a ruminant alphaherpesvirus. A viral isolate obtained from the genital mucosa of a female red deer was characterised by sequencing and restriction endonuclease analyses as a cervid herpesvirus 1 closely related to Scottish Banffshire 82 strain.

We evaluated the management of genital herpes simplex virus (HSV) infection at our clinic. Over six months, 135 patients with primary HSV and 97 patients with recurrent HSV attended. Sixty percent of patients presenting within five days of primary infection were treated with oral acyclovir. Only 53% of patients with primary HSV were given written/verbal information. Follow-up appointments were attended by 78%. Seventy-seven percent of patients with recurrent HSV had at least one viral isolate typed and 72% of patients experiencing episodes exceeding, annually, six were offered suppressive therapy. Our results demonstrate successful management of patients with HSV infections but recommendations are made to reach the standards set by BASSH. Furthermore, we illustrate that improved documentation would go a long way in validating the result.

We describe a case of fatal neonatal enteroviral infection caused by echovirus 7. Multiple treatments, including specific antiviral therapy, were attempted and failed. Studies of the viral isolate, maternal plasma, intravenous immunoglobulin preparations and pleconaril were performed to identify potential causes for treatment failure.

With input from the gene therapy community, CBER is actively examining the recommendations for RCR testing during retroviral vector production, production of ex vivo-transduced cells, and in patients who receive such material. Our initial recommendations were made at a time when our experience with RCR detection assays and clinical use of retroviral vectors was limited. As the gene therapy field has matured, there is an increasing amount of data available on RCR detection assays and from monitoring of patients in clinical trials. The cumulative data give assurance that RCR detection assays in use are of sufficient sensitivity to provide a margin of safety to patients: no patients to date have evidence of RCR infection. However, CBER encourages members of the gene therapy community to continue to submit data to the FDA or to publish data that will enhance the cumulative data base on RCR testing assays, experience with different VPC, and patient monitoring. Based on the analysis of data accumulated to data, and ongoing discussions with members of the gene therapy community, CBER is proposing to discuss changes to the current RCR testing recommendations, as summarized below. RCR testing during production of retroviral vector and ex vivo-transduced cells. Development of characterized standards for RCR testing of supernatant and cells should allow comparison of assay sensitivity. One proposal under consideration is to apply statistical methods to determine how much material needs to be tested independent of the size of the production lot. Data and discussion are still needed to define a limit concentration and a value for probability of detection for RCR testing, while maintaining an appropriate margin of safety. These modifications of RCR testing strategies could lead to improvements in assay sensitivity. Additional discussion and data are also needed to evaluate the current recommendations of the testing for ex vivo-transduced cells: should both cells and supernatant be tested in all cases? RCR testing during patient follow-up. The time points required for RCR testing during patient follow-up need examination. One proposal under consideration is to sample and assay at three time points during the first year of treatment (e.g., 4-6 weeks, 3 months, and 1 year post-treatment). Further discussion is needed to define appropriate additional follow-up. Choice of assays to detect surrogate markers for RCR infection (i.e., serologic or PCR-based assays) should consider mode of vector administration and the patient population. Positive results with such assays should be pursued by direct culture assay to obtain and characterize the infectious viral isolate. These proposals will be the focal point for the discussion at the Retroviral Vector Breakout Session at the 1997 FDA/NIH Gene Therapy Conference. After the 1997 FDA/NIH Gene Therapy Conference, CBR plans to propose revised recommendations for RCR testing for public comment.

Simian immunodeficiency virus (SIV) is a designation for a group of related but unique lentiviruses identified in several primate species. A viral isolate from a rhesus macaque (i.e., SIVmac) causes a fatal AIDS-like disease in experimentally infected macaques, and several infectious molecular clones of this virus have been characterized. This report presents the complete nucleotide sequence of molecularly cloned SIVmac1A11, and comparisons are made with the sequence of molecularly cloned SIVmac239. SIVmac1A11 has delayed replication kinetics in lymphoid cells but replicates as well as uncloned SIVmac in macrophage cultures. Macaques infected with virus from the SIVmac1A11 clone develop antiviral antibodies, but virus does not persist in peripheral blood mononuclear cells and no disease signs are observed. SIVmac239 infects lymphoid cells, shows restricted replication in cultured macrophages, and establishes a persistent infection in animals that leads to a fatal AIDS-like disease. Both viruses are about 98% homologous at the nucleotide sequence level. In SIVmac1A11, the vpr gene as well as the transmembrane domain of env are prematurely truncated, whereas the nef gene of SIVmac239 is prematurely truncated. Sequence differences are also noted in variable region 1 (V1) in the surface domain of the env gene. The potential implications of these and other sequence differences are discussed with respect to the phenotypes of both viruses. This animal model is critically important for investigating the roles of specific viral genes in viral/host interactions that cannot be studied in individuals infected with the human immunodeficiency virus (HIV).

The biochemical and biophysical characteristics of the closely related Diacrisia virginica and Hyphantria cunea granulosis virus isolates were examined. Sucrose gradient sedimentation patterns of alkali-solubilized DGV and HcGV capsules were identical. The top, middle, and bottom fractions from either viral isolate were infectious when injected into susceptible host larvae. Electrophoretic analysis of alkaline-solubilized granulin extracts demonstrated that both viruses contain alkaline proteolytic activity. The major granulin protein (∼28,000 daltons) of both isolates comigrated in a SDS-PAGE. Electrophoretic separation of the virus proteins demonstrated some quantitative differences between the two granulosis viruses. The enveloped nucleocapsids and the nucleocapsids of the two viruses were morphologically indistinguishable.

Two Egyptian male patients with sand fly fever-Naples virus infection are presented. The virus was isolated from one patient while both patients had diagnostic rises in indirect fluorescent antibody titers to the virus. The viral isolate, SFN 85055, grows to much higher titers and plaques more efficiently than the prototype sand fly fever-Naples virus and should facilitate work with this virus.

A virus has been isolated from the corneal epithelium of a patient with superficial punctate keratitis. This virus has been identified as varicella-zoster. The patient's serum had a low titer of neutralizing antibodies to varicella-zoster virus. The significance of this finding is uncertain, but possible mechanisms involved in the pathogenesis of this condition are discussed.

Three cases of skin ulceration in cats are described. Herpesvirus was isolated from the ulcers. Treatment of one case with antibiotics and an antiviral agent was successful. The significance of the viral isolate is discussed.

Publisher Summary This chapter focuses on the immunological techniques used in the study of animal viruses. The basis for the immunological techniques employed in the study of animal viruses is the same as for all other microorganisms— an antigen–antibody reaction. The chapter discusses the neutralization test, complement–fixation (CF) test, fluorescent-antibody (FA) techniques and hemagglutination and hemagglutination-inhibition (HI) tests. The neutralization test is used with the animal viruses longer than other serological tests, and its result is generally taken as the final, decisive criterion for identification of the serotype of a viral isolate. It is considered to be the most universally specific of the various tests in use. HI test is used for the identification of new isolates and for the determination of the presence of antibodies, either as an aid to the specific diagnosis of a current illness or for surveys. With some viruses, antibodies detected by HI are similar in specificity to those found by neutralization test. The CF test is extensively used is in the diagnosis of a large number of viral infections of man. The test is useful as an aid to the diagnosis because by testing two specimens of serum, one taken early in the acute phase of the disease and the other 2 or 3 weeks later, it is possible to detect a significant rise in the titer of antibody, 4-fold or greater, more often than with other serological tests. An added reason for the use of the CF test is that it is relatively simple to perform, technically dependable, and well adjusted to large numbers of specimens.

On 10 September 2011, a patient in his 50s was admitted to hospital in Ancona, Italy, after six days of high fever and no response to antibiotics. West Nile virus (WNV) infection was suspected after tests to determine the aetiology of the fever were inconclusive. On 20 September, WNV-specific IgM and IgG antibodies were detected in the patient's serum. Genomic sequencing of the viral isolate showed that the virus belonged to WNV lineage 2. .

Encephalomyocarditis virus was isolated from the organs of swine dying during an outbreak of an acutely fatal disease occurring on a farm in Panama. The outstanding lesion was severe myocarditis. Pigs inoculated with the viral isolate developed a systemic infection with myocarditis.

In Reply.— Drs Karjalainen and Heikkila have emphasized the frequent similarity between acute myopericarditis and myocardial infarction. Our case report of myocardial infarction in a highly conditioned swimmer also considered the possibility of myopericarditis. However, the absence of preceding symptoms of viral illness and the highly localized uptake of technetium Tc 99m pyrophosphate in the anterolateral wall argued against myopericarditis. We did not perform viral serological studies because a primary viral isolate was not recovered from the throat. A limited number of radionuclide studies in documented viral myopericarditis have shown predominantly diffuse uptake patterns 1 with a single report of localized radionuclide uptake. 2 Each of these cases had definite preceding symptoms of viral illness. We agree that myopericarditis may mimick acute myocardial infarction. We also emphasize that myocardial infarction is well documented in young adults with normal coronary arteries. 3 In some of these cases, exercise is a precipitating

A gravid woman with herpes Type II encephalitis delivered an infant with herpes neonatorum despite therapy with acyclovir. Acyclovir was not measurable in the baby's serum 10 h after birth. The viral isolate was sensitive to acyclovir in vitro, and the neonatal infection responded to treatment with the drug. Prenatal antiviral therapy may be ineffective in preventing intrauterine herpesvirus infection.

Echovirus 14 was isolated from the liver of a newborn infant with fatal hepatic necrosis. It appeared likely that the infection was acquired in utero. Ultrastructurally, the findings in the liver cells were consistent with a viral infection. In cell cultures the viral isolate was found to produce crystalline inclusions in the nucleus and cytoplasm. Negative stained virions measured approximately 30.3 mμ in diameter. To our knowledge, the electron microscopy of echovirus 14 has not been previously reported.