Sequence and phylogenetic analysis of highly pathogenic avian influenza H5N1 viruses isolated during 2006-2008 outbreaks in Pakistan reveals genetic diversity.

BACKGROUND: Since the first outbreak recorded in northern areas of Pakistan in early 2006, highly pathogenic avian influenza H5N1 viruses were isolated from commercial poultry and wild/domestic birds from different areas of Pakistan up to July 2008. Different isolates of H5N1 were sequenced to explore the genetic diversity of these viruses. RESULTS: Phylogenetic analysis revealed close clustering and highest sequence identity in all 8 genes to HPAI H5N1 isolates belonging to unified H5 clade 2.2, sub-lineage EMA-3 recovered from Afghanistan during the same time period. Two subgroups within Pakistani H5N1 viruses, from domestic and wild birds, were observed on the basis of their sequence homology and mutations. HPAI motif, preferred receptor specificity for α-(2, 3) linkages, potential N-linked glycosylation sites and an additional glycosylation site at the globular head of HA protein of four Pakistani H5N1 isolates. While, the amino acids associated with sensitivities to various antiviral drugs (Oseltamivir, Zanamivir, Amantadine) were found conserved for the Pakistani H5N1 isolates. Conspicuously, some important mutations observed at critical positions of antigenic sites (S141P, D155S, R162I & P181S) and at receptor binding pocket (A185T, R189K & S217P) of HA-1. A high sequence similarity between Pakistani HP H5N1 and LP H9N2 viruses was also observed. Avian like host specific markers with the exception of E627K in PB2, K356R in PA, V33I in NP, I28V in M2 and L107F in NS2 proteins were also observed. CONCLUSIONS: Various point mutations in different genes of H5 viruses from Pakistan were observed during its circulation in the field. The outbreaks started in Khyber Pakhtoon Khawa (North West) province in 2006 and spread to the Southern regions over a period of time. Though migratory birds may have a role for this continued endemicity of clade 2.2 H5N1 viruses during 2006-2008 in Pakistan, the possibility of their transmission through legal or illegal poultry trade across the borders cannot be ignored.

Multiple Genome Sequences of Foot-and-Mouth Disease Virus Asia-1 Lineage Sindh-08 from Outbreaks in Pakistan, 2011 to 2012.

We report the near-full-length genome sequences of 22 isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1, lineage Sindh-08, obtained from foot-and-mouth disease outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these new genomes for understanding the regional molecular epidemiology.

Multiple Genomes of Foot-and-Mouth Disease Virus Serotype Asia-1 Obtained from Subclinically Infected Asian Buffalo (Bubalus bubalis) in Pakistan.

We report the near-full-genome sequences of 49 isolates of serotype Asia-1 foot-and-mouth disease virus obtained from subclinically infected Asian buffalo in Islamabad Capital Region, Pakistan, in 2011 to 2012. Sequences from subclinically infected animals are exceedingly rare and complement the more commonly available sequences acquired from clinical cases.

Foot-and-Mouth Disease Virus Serotypes O and A from Outbreaks in Pakistan 2011-2012.

We report the near full genome sequences of 18 isolates of foot-and-mouth disease virus serotype O and 6 isolates of serotype A obtained from outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these genomes for understanding regional molecular epidemiology.

Genome Sequences of Foot-and-Mouth Disease Virus Serotype A and O Strains Obtained from Subclinically Infected Asian Buffalo (Bubalus bubalis) in Pakistan.

We report the nearly full genome sequences of 14 isolates of serotype A foot-and-mouth disease virus and 5 isolates of serotype O, which were obtained from subclinically infected Asian buffalo in Pakistan in 2011 to 2012. Sequences from subclinically infected animals are rare and complement the more commonly available sequences from clinical cases.

A novel augmented deep transfer learning for classification of COVID-19 and other thoracic diseases from X-rays.

Deep learning has provided numerous breakthroughs in natural imaging tasks. However, its successful application to medical images is severely handicapped with the limited amount of annotated training data. Transfer learning is commonly adopted for the medical imaging tasks. However, a large covariant shift between the source domain of natural images and target domain of medical images results in poor transfer learning. Moreover, scarcity of annotated data for the medical imaging tasks causes further problems for effective transfer learning. To address these problems, we develop an augmented ensemble transfer learning technique that leads to significant performance gain over the conventional transfer learning. Our technique uses an ensemble of deep learning models, where the architecture of each network is modified with extra layers to account for dimensionality change between the images of source and target data domains. Moreover, the model is hierarchically tuned to the target domain with augmented training data. Along with the network ensemble, we also utilize an ensemble of dictionaries that are based on features extracted from the augmented models. The dictionary ensemble provides an additional performance boost to our method. We first establish the effectiveness of our technique with the challenging ChestXray-14 radiography data set. Our experimental results show more than 50% reduction in the error rate with our method as compared to the baseline transfer learning technique. We then apply our technique to a recent COVID-19 data set for binary and multi-class classification tasks. Our technique achieves 99.49% accuracy for the binary classification, and 99.24% for multi-class classification.

Sequence and phylogenetic analysis of H7N3 avian influenza viruses isolated from poultry in Pakistan 1995-2004.

BACKGROUND: Avian influenza virus (AIV) infections have caused heavy economic losses to the poultry industry in Pakistan as well as numerous other regions worldwide. The first introduction of H7N3 AIV to Pakistan occurred during 1995, since then H7N3, H9N2 and H5N1 AIVs have each been sporadically isolated. This report evaluates the genetic origin of the H7N3 viruses from Pakistan collected 1995-2004 and how they disseminated within the country. To accomplish this we produced whole genome sequences for 6 H7N3 viruses and data for the HA and NA genes of an additional 7 isolates. All available sequence from H7N3 AIV from Pakistan was included in the analysis. RESULTS: Phylogenetic analysis revealed that there were two introductions of H7 into Pakistan and one N3 introduction. Only one of the H7 introductions appears to have become established in poultry in Pakistan, while the other was isolated from two separate outbreaks 6 years apart. The data also shows that reassortment has occurred between H7N3 and H9N2 viruses in the field, likely during co-infection of poultry. Also, with the exception of these few reassortant isolates, all 8 genes in the predominant H7N3 virus lineage have evolved to be phylogenetically distinct. CONCLUSIONS: Although rigorous control measures have been implemented in commercial poultry in Pakistan, AIV is sporadically transmitted to poultry and among the different poultry industry compartments (broilers, broiler breeders, table egg layers). Since there is one primary H7 lineage which persists and that has reassorted with the H9N2 AIV in poultry, it suggests that there is a reservoir with some link commercial poultry. On a general level, this offers insight into the molecular ecology of AIV in poultry where the virus has persisted despite vaccination and biosecurity. This data also illustrates the importance of sustained surveillance for AIVs in poultry.

Passive immunization against highly pathogenic Avian Influenza Virus (AIV) strain H7N3 with antiserum generated from viral polypeptides protect poultry birds from lethal viral infection.

Our studies were aimed at developing a vaccination strategy that could provide protection against highly pathogenic avian influenza virus (AIV), H7N3 or its variants outbreaks. A purified viral stock of highly pathogenic H7N3 isolate was lysed to isolate viral proteins by electrophresing on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by their elution from gel through trituration in phosphate buffered saline (PBS). Overall, five isolated viral polypeptides/proteins upon characterization were used to prepare hyperimmune monovalent serum against respective polypeptides independently and a mixture of all five in poultry birds, and specificity confirmation of each antiserum through dot blot and Western blotting. Antiserum generated from various group birds was pooled and evaluated in 2-week old broiler chicken, for its protection against viral challenge. To evaluate in-vivo protection of each antiserum against viral challenges, six groups of 2-week old broiler chicken were injected with antiserum and a seventh control group received normal saline. Each group was exposed to purified highly pathogenic AIV H7N3 strain at a dose 10(5) embryo lethal dose (ELD(50)). We observed that nucleoprotein (NP) antiserum significantly protected birds from viral infection induced morbidity, mortality and lowered viral shedding compared with antiserum from individual viral proteins or mixed polypeptides/proteins inclusive of NP component. The capability of individual viral polypeptide specific antisera to protect against viral challenges in decreasing order was nucleoprotein (NP) > hemagglutinin (HA) > neuraminidase (NA) > viral proteins mix > viral polymerase (PM) > non-structural proteins (NS). Our data provide proof of concept for potential utilization of passive immunization in protecting poultry industry during infection outbreaks. Furthermore conserved nature of avian NP makes it an ideal candidate to produce antiserum protective against viral infection.

Genetic stability of foot-and-mouth disease virus during long-term infections in natural hosts.

Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008-2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008-2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.

Characterization of naturally occurring, new and persistent subclinical foot-and-mouth disease virus infection in vaccinated Asian buffalo in Islamabad Capital Territory, Pakistan.

The convalescent subclinical carrier state of foot-and-mouth disease virus (FMDV) infection has been thoroughly investigated; contrastingly, the subclinical form of new infections of vaccinated and naïve hosts is recognized, but poorly understood. To investigate the natural dynamics of subclinical FMDV infections, a prospective, 12-month, longitudinal study was conducted in vaccinated Asian buffalo (Bubalus bubalis) under natural conditions in Pakistan, where FMDV is hyperendemic. Oropharyngeal fluid (OPF) samples were obtained quarterly from 300 buffalo on 30 farms which reported no clinical FMD during the 12-month study period. At the start of the study, 77.7% of buffalo had FMDV anti-NSP antibodies, and all farms had at least one seropositive buffalo. Based upon the presence of viral RNA and viral VP1 sequences obtained, distinct subcategories of subclinical infections were documented, including new, persistent, and serial infections with different FMDV strains. Viral RNA was detected in at least one OPF sample from 180 (60%) of the 300 buffalo. Over the course of the study, FMDV was detected in OPF of 80 buffalo that had been FMDV-free in previous OPF samples, indicating the occurrence of new subclinical infections. Eight buffalo were confirmed to be persistently infected, and serial infection with different FMDVs was confirmed in 13 animals. The most prevalent serotype detected was Asia-1, followed by A, and O. Phylogenetic analysis indicated multiple distinct clusters of serotypes Asia-1 and A. This study indicates a high prevalence of subclinical FMDV infection in vaccinated buffalo in Pakistan and emphasizes the importance of clinically undetected infection in FMD dynamics in endemic regions.

One Health research and training and government support for One Health in South Asia.

INTRODUCTION: Considerable advocacy, funding, training, and technical support have been provided to South Asian countries to strengthen One Health (OH) collaborative approaches for controlling diseases with global human pandemic potential since the early 2000s. It is essential that the OH approach continues to be strengthened given South Asia is a hot spot for emerging and endemic zoonotic diseases. The objectives of this article are to describe OH research and training and capacity building activities and the important developments in government support for OH in these countries to identify current achievements and gaps. MATERIALS AND METHODS: A landscape analysis of OH research, training, and government support in South Asia was generated by searching peer-reviewed and grey literature for OH research publications and reports, a questionnaire survey of people potentially engaged in OH research in South Asia and the authors' professional networks. RESULTS: Only a small proportion of zoonotic disease research conducted in South Asia can be described as truly OH, with a significant lack of OH policy-relevant research. A small number of multisectoral OH research and OH capacity building programmes were conducted in the region. The governments of Bangladesh and Bhutan have established operational OH strategies, with variable progress institutionalising OH in other countries. Identified gaps were a lack of useful scientific information and of a collaborative culture for formulating and implementing integrated zoonotic disease control policies and the need for ongoing support for transdisciplinary OH research and policy-relevant capacity building programmes. DISCUSSION: Overall we found a very small number of truly OH research and capacity building programmes in South Asia. Even though significant progress has been made in institutionalising OH in some South Asian countries, further behavioural, attitudinal, and institutional changes are required to strengthen OH research and training and implementation of sustainably effective integrated zoonotic disease control policies.

Rouleaux formation of white blood cells and platelets in leukemia / Formação de rouleaux de glóbulos brancos e plaquetas em leucemia

The objective of this study was to measure the effects of glucose and salt level on white blood cells, red blood cells and platelets (PLTs) in the blood of a leukemic patient by using a white light microscope. Different concentrations of glucose and salt in the range of 0 mM to 500 mM were admixed in the blood sample to prepare blood smear. We revealed that shape of erythrocytes, leukocytes and platelets changes and form aggregates. Increasing concentrations of glucose cause to increases aggregation process of white blood cells, red blood cells and platelets. And the increasing concentration of sodium chloride causes to increase rouleaux formation and aggregation of platelets but dehydration due to increased sodium chloride concentration causes to break the aggregation of white blood cells. Comparison of CBC reports of these samples with and without analytes shows that total leukocyte count (TLC) decreases gradually towards normal ranges of leukocytes which is favorable in the treatment of leukemia but at the same time decreasing level of hemoglobin HGB, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) and increasing level of red blood cell (RBCs) causes to reduce oxygen supply which is in favor of cancer growth and anemia. This work provides us the base for translation this in vitro study towards the in vivo case of blood microvasculature as a non-invasive methodology.

O objetivo deste estudo foi medir os efeitos da glicose e do nível de sal nos glóbulos brancos, glóbulos vermelhos e plaquetas (PLTs) no sangue de um paciente leucêmico usando um microscópio de luz branca. Foram misturadas diferentes concentrações de glicose e sal na gama de 0 mM a 500 mM na amostra de sangue para preparar esfregaço de sangue. Descrevemos que a forma dos eritrócitos, leucócitos e plaquetas muda e forma agregados. O aumento das concentrações de glicose aumenta o processo de agregação de glóbulos brancos, glóbulos vermelhos e plaquetas. E a crescente concentração de cloreto de sódio causa o aumento da formação de rouleaux e a agregação de plaquetas, mas a desidratação devido ao aumento da concentração de cloreto de sódio causa a quebra da agregação de glóbulos brancos. A comparação dos relatórios de CBC dessas amostras com e sem analitos mostra que a contagem total de leucócitos (TLC) diminui gradualmente para os intervalos normais de leucócitos, o que é favorável no tratamento da leucemia, mas ao mesmo tempo diminui o nível de hemoglobina HGB, hemoglobina corpuscular média (MCH ), a concentração média de hemoglobina corpuscular (MCHC) e o aumento do nível de glóbulos vermelhos (RBCs) reduz o suprimento de oxigênio, o que é a favor do crescimento do câncer e da anemia. Este trabalho fornece a base para a tradução deste estudo in vitro para o caso in vivo de microvasculatura de sangue como uma metodologia não-invasiva.

Sequence and phylogenetic analysis of virulent Newcastle disease virus isolates from Pakistan during 2009-2013 reveals circulation of new sub genotype.

Despite observing the standard bio-security measures at commercial poultry farms and extensive use of Newcastle disease vaccines, a new genotype VII-f of Newcastle disease virus (NDV) got introduced in Pakistan during 2011. In this regard 300 ND outbreaks recorded so far have resulted into huge losses of approximately USD 200 million during 2011-2013. A total of 33 NDV isolates recovered during 2009-2013 throughout Pakistan were characterized biologically and phylogenetically. The phylogenetic analysis revealed a new velogenic sub genotype VII-f circulating in commercial and domestic poultry along with the earlier reported sub genotype VII-b. Partial sequencing of Fusion gene revealed two types of cleavage site motifs; lentogenic (112)GRQGRL(117) and velogenic (112)RRQKRF(117) along with some point mutations indicative of genetic diversity. We report here a new sub genotype of virulent NDV circulating in commercial and backyard poultry in Pakistan and provide evidence for the possible genetic diversity which may be causing new NDV out breaks.

H7 avian influenza virus vaccines protect chickens against challenge with antigenically diverse isolates.

Vaccination has been a critical tool in the control of some avian influenza viruses (AIV) and has been used routinely in Pakistan to help control sporadic outbreaks of highly pathogenic (HP) H7 AIV since 1995. During that time, several AIV isolates were utilized as inactivated vaccines with varying degrees of success. In order to evaluate which H7 AIV strains may serve as optimal vaccines for diverse H7 AIVs from Pakistan we conducted vaccination-challenge studies with five H7 vaccines against challenge with two HPAIVs: A/chicken/Murree/NARC-1/1995 H7N3 and A/chicken/Karachi/SPVC-4/2004 H7N3. To further characterize the isolates antigenic cartography was used to visually demonstrate the antigenic relationships among the isolates. All vaccines provided similar protection against mortality, morbidity and shedding of challenge virus from the respiratory tract. However, some minor (not statistically significant) differences were observed and correlated with antibody levels induced by the vaccine prior to challenge.

Zoonotic potential of highly pathogenic avian H7N3 influenza viruses from Pakistan.

H5 and H7 avian influenza viruses can become highly pathogenic in chickens after interspecies transmission. These viruses have transmitted directly to humans from birds in Eurasia and Africa (H5N1), the Netherlands (H7N7), and Canada (H7N3). Here we report antigenic, sequence, and phylogenetic analyses of H7N3 viruses isolated from chickens in Pakistan from 1995 to 2002. We compared the pathogenic and zoonotic potential of the Pakistani viruses in avian and mammalian hosts. In chickens, all of the isolates showed high pathogenicity with poor transmissibility to contact birds. Viral shedding from the trachea and cloaca was equivalent, but cloacal shedding occurred longer; dissemination of virus into the tissues was widespread. In contrast, the viruses replicated poorly in 6-week-old mallard ducks. In mammalian hosts, of the two Pakistani H7N3/02 viruses that caused weight loss, one also caused 40% mortality in mice without prior adaptation, and preliminary experiments in ferrets showed significant virus multiplication in the lungs, intestine, and conjunctiva. We conclude that the H7N3/02 isolates from Pakistan show limited antigenic drift and have evolved slowly during their 8-year circulation in chickens; however, these viruses have the potential to infect mammals.